Methods of treating and diagnosing disease using biomarkers for bcg therapy

ABSTRACT

The invention provides methods of treating disease, such as hypercholesterolemia (e.g., by modulating serum lipids, such as cholesterol, low-density lipoproteins, high-density lipoproteins, and triglycerides) and hyperglycemia by administering  Bacillus  Calmette-Guerin (BCG). Methods of the invention also encompass the use of genomic, proteomic, and metabolomic analyses for determining the likelihood that a patient has disease or will respond to treatment (e.g., with BCG therapy), as well as for determining whether a patient previously administered BCG would benefit from BCG redosing.

FIELD OF THE INVENTION

The invention relates to methods of treating diseases, such ashypercholesterolemia, by administering Bacillus Calmette-Guerin (BCG),as well as methods for diagnosing a subject as having disease or needingtreatment, e.g., with BCG, based on genomic, proteomic, and metabolomicanalyses.

BACKGROUND OF THE INVENTION

Elevated levels of cholesterol and other serum lipids, such aslow-density lipoproteins (LDLs) and triglycerides, represent a prominentthreat to human health and have been correlated with the onset of avariety of diseases. Among the conditions associated with highcholesterol are heart disease and stroke, the pathology of each of whichis characterized by a reduction in the circulation of blood to vitalbodily organs due to resistance imposed on the flow of blood throughhardened arteries. According to the World Health Organization,approximately a third of ischemic heart disease cases worldwide areattributable to high cholesterol. Diseases associated with high serumcholesterol levels remain challenging indications for conventionaltherapies, and there is currently a need for new therapeutic modalitiesfor restoring blood serum lipid levels to within a healthy range.

SUMMARY OF THE INVENTION

The present invention provides methods for modulating serum lipidlevels, such as the levels of cholesterol, LDLs, HDLs, andtriglycerides, by administration of BCG (e.g., Mycobacterium bovis), toa subject (e.g., a mammalian subject, such as a human). The inventionalso features methods for preventing the onset of elevated cholesterol,LDL, and triglyceride levels by administering BCG to a subject prone tothe development of elevated levels of these serum lipids, as well asmethods of treating and preventing a wide range of diseases associatedwith the accumulation of these substances. The invention additionallyprovides methods for diagnosing various diseases in a subject, such asimmunological and neurological disorders, as well as pathologiesassociated with heightened levels of cholesterol, LDLs, and/ortriglycerides, by assessing the presence, amount, or concentration ofone or more biomarkers in a subject. The diagnostic methods describedherein can be used not only to determine whether a subject has aparticular disease or condition, but also to evaluate the likelihoodthat the subject will respond to treatment with a therapeutic agent(e.g., BCG) or will benefit from treatment with one or more additionaldoses of a therapeutic agent (e.g., BCG).

In a first aspect, the invention features a method of reducing the levelof cholesterol, LDL, or triglycerides in a subject in need thereof byadministering an effective amount of BCG to the subject. In anotheraspect, the invention relates to a method of increasing the level of HDLin a subject in need thereof by administering an effective amount of BCGto the subject. In some embodiments of the invention, the administrationof BCG lowers the level of total cholesterol in the subject by 1%, 5%,or more. In some cases, the subject has a total cholesterol level ofabout 100 mg/dL or greater, such as a total cholesterol level of about129 mg/dL or greater. Additionally or alternatively, the administrationof BCG may lower the level of LDLs in the subject by 1%, 5%, or more. Insome cases, the subject has a LDL level of about 80 mg/dL or greater. Incertain embodiments, the administration of BCG elevates the level ofHDLs in the subject by 1%, 5%, or more. In particular cases, the subjecthas a HDL level of about 40 mg/dL or below. Additionally oralternatively, the subject may have a triglyceride level of about 100mg/dL or greater. In certain embodiments, the subject has a triglyceridelevel of between about 100 mg/dL and about 500 mg/dL and/or a ratio oftotal cholesterol level to HDL level of about 5 or greater. In otherembodiments, the subject has a ratio of total cholesterol level to HDLlevel of between about 3 and about 10. In some embodiments, theinvention features methods of stabilizing the level of cholesterol, LDL,or triglycerides in a subject, for instance, by administration of BCG asdescribed herein. In some embodiments, the invention features methods ofpreventing an increase in the level of cholesterol, LDL, ortriglycerides in a subject, for instance, by administration of BCG asdescribed herein. In some embodiments, the invention features methods ofstabilizing the level of HDL in a subject, for instance, byadministration of BCG as described herein. In some embodiments, theinvention features methods of preventing a decrease in the level of HDL,for instance, by administration of BCG as described herein.

The methods of the invention can also be applied to a subject that has atotal cholesterol, LDL and/or triglyceride level that is higher thanthat which has previously been observed for the subject. For instance,the subject may have previously been observed as having a totalcholesterol level less than about 180 mg/dL. In some cases, the subjectmay have previously been observed as having a LDL level of less thanabout 100 mg/dL and/or a triglyceride level of less than about 150mg/dL.

In embodiments of any of the above-described aspects of the invention,the subject may be suffering from a disease associated with an elevatedlevel of cholesterol, such as hypercholesterolemia, hyperlipidemia,coronary heart disease, peripheral arterial disease (PAD), peripheralvascular disease, hypertension, stroke, diabetes, metabolic syndrome,obesity, or insulin resistance. BCG may be administered to the subjectin an amount sufficient to alleviate or reduce a symptom associated withthe disease. For instance, the symptom may be an elevated level of asubstance such as lactate dehydrogenase (LDH), LDL, or triglycerides.The administration of BCG may serve to reduce the level of one or moreof these substances. In other embodiments, the symptom may be angina,arrhythmia, and heart failure. In these cases, the administration of BCGmay alleviate or reduce the angina, arrhythmia, or heart failure.

In some embodiments of the invention, BCG is administered to a subjectin conjunction with a hypolipidemic agent, such as a HMG-CoA reductaseinhibitor (e.g., atorvastatin, cerivastatin, fluvastatin, lovastatin,mevastatin, pitavastatin, pravastatin, rosuvastatin, simvastatin, orcombinations thereof), niacin, a fibric acid derivative (e.g.,fenofibrate or gemfibrozil), a cholesterol absorption inhibitor (e.g.,ezetimibe), and/or a lipolytic agent (e.g., norepinephrine,isoproterenol, forskolin, bucladesine, or theophylline). In some cases,BCG is admixed with the hypolipidemic agent (e.g., in a singlepharmaceutical composition), while in other cases, BCG is administeredseparately from the hypolipidemic agent (e.g., consecutively and/or by adifferent route of administration).

In another aspect, the invention provides a method of treating a subjectthat has a disease, such as an autoimmune disease or neurologicalcondition as described herein, by administering BCG to the subject. Insome embodiments, the subject has been previously diagnosed as havingthe disease, e.g., using methods known in the art.

In an additional aspect, the invention features a method of treating asubject that has previously been diagnosed as having a disease, such asan autoimmune disease or a neurological condition described herein, suchthat the subject has been diagnosed by:

-   -   a) determining a quantity of methylated cytosine residues in a        sample of nuclear DNA isolated from the subject; and    -   b) comparing the quantity to a quantity of methylated cytosine        residues in a reference sample (e.g., a sample isolated from a        control subject not having the disease, such as a subject of the        same age, gender, and/or weight),

such that a determination that the quantity of methylated cytosineresidues in the sample of nuclear DNA isolated from the subject isgreater than the quantity of methylated cytosine residues in thereference sample indicates that the subject has the disease, the methodhaving the step of administering BCG to the subject. In other aspects ofthe invention, a determination that the quantity of methylated cytosineresidues in the sample of nuclear DNA isolated from the subject is lessthan the quantity of methylated cytosine residues in the referencesample indicates that the subject has the disease, and the methodfurther includes administering an effective amount of BCG to thesubject.

In another aspect, the invention provides a method of treating a subjectthat has previously been diagnosed as having a disease such as anautoimmune disease or a neurological condition, such that the subjecthas been diagnosed by:

-   -   a) i) determining a level of one or more substances selected        from the group consisting of an mRNA molecule encoding a        cytokine or lipolytic protein, a protein selected from the group        consisting of a cytokine and a lipolytic protein, an acetylated        amino acid selected from the group consisting of        N-acetylalanine, N-acetylaspartic acid, N-acetylserine,        N-acetylthreonine, N-acetylhistidine,        N-acetyl-3-methylhistidine, N-acetylvaline, and        N-α-acetyllysine, and N-acetylmethionine, or a methylated        metabolite selected from the group consisting of        N-α-acetyl-3-methylhistidine, 3-methylglutaconic acid,        3-methylglutarylcarnitine, and N-ε-trimethyllysine in a sample        from the subject; and        -   (ii) comparing the level of the one or more substances to            the level of the one or more substances in a reference            sample (e.g., a sample isolated from a control subject not            having the disease, such as a subject of the same age,            gender, and/or weight), such that a determination that the            level of the one or more substances in the sample from the            subject having the disease is less than the level of the one            or more substances in the reference sample indicates that            the subject has the disease; or    -   b) (i) determining a level of one or more substances selected        from the group consisting of an mRNA molecule encoding a        lipogenic protein, an adiponectin receptor, a lysine        acetyltransferase, a histone acetyltransferase, a histone        deacetylase, or a histone, a protein selected form the group        consisting of a lipogenic protein, an adiponectin receptor, a        lysine acetyltransferase, a histone acetyltransferase, a histone        deacetylase, and a histone, or a methylated metabolite selected        from the group consisting of 4-methyl-2-oxopentanoic acid and        3-methyl-2-oxobutyric acid in a sample from the subject; and        -   (ii) comparing the level of the one or more substances to            the level of the one or more substances in a reference            sample (e.g., a sample isolated from a control subject not            having the disease, such as a subject of the same age,            gender, and/or weight), such that a determination that the            level of the one or more substances in the sample from the            subject having the disease is greater than the level of the            one or more substances in the reference sample indicates            that the subject has the disease, such that the method            includes administering an effective amount of BCG to the            subject.

In some cases, the autoimmune disease described above is type Idiabetes, Alopecia Areata, Ankylosing Spondylitis, AntiphospholipidSyndrome, Autoimmune Addison's Disease, Autoimmune Hemolytic Anemia,Autoimmune Hepatitis, Behcet's Disease, Bullous Pemphigoid,Cardiomyopathy, Celiac Sprue-Dermatitis, Chronic Fatigue ImmuneDysfunction Syndrome (CFIDS), Chronic Inflammatory DemyelinatingPolyneuropathy, Churg-Strauss Syndrome, Cicatricial Pemphigoid, CRESTSyndrome, Cold Agglutinin Disease, Crohn's Disease, Essential MixedCryoglobulinemia, Fibromyalgia-Fibromyositis, Graves' Disease,Guillain-Barré, Hashimoto's Thyroiditis, Hypothyroidism, IdiopathicPulmonary Fibrosis, Idiopathic Thrombocytopenia Purpura (ITP), IgANephropathy, Juvenile Arthritis, Lichen Planus, Lupus, Ménière'sDisease, Mixed Connective Tissue Disease, Multiple Sclerosis, MyastheniaGravis, Pemphigus Vulgaris, Pernicious Anemia, Polyarteritis Nodosa,Polychondritis, Polyglandular Syndromes, Polymyalgia Rheumatica,Polymyositis and Dermatomyositis, Primary Agammaglobulinemia, PrimaryBiliary Cirrhosis, Psoriasis, Raynaud's Phenomenon, Reiter's Syndrome,Rheumatic Fever, Rheumatoid Arthritis, Sarcoidosis, Scleroderma,Sjögren's Syndrome, Stiff-Man Syndrome, Takayasu Arteritis, TemporalArteritis/Giant Cell Arteritis, Ulcerative Colitis, Uveitis, Vasculitis,Vitiligo, or Wegener's Granulomatosis.

In certain cases, the neurological condition described above is a braintumor, a brain metastasis, a spinal cord injury, schizophrenia,epilepsy, Amyotrophic lateral sclerosis (ALS), Parkinson's disease,Autism, Alzheimer's disease, Huntington's disease, or stroke.

In an additional aspect, the invention provides a method of treating asubject having a disease selected from the group consisting of a braintumor, a brain metastasis, schizophrenia, epilepsy, Autism, stroke, anallergy, allograft rejection, graft-versus-host disease, asthma, maculardegeneration, muscular atrophy, a disease related to miscarriage,atherosclerosis, bone loss, a musculoskeletal disease, and obesity, byadministering an effective amount of BCG to the subject. In someembodiments of the invention, the subject has been previously diagnosedas having the disease (e.g., using methods known in the art or byanalysis of one or more biomarkers described herein, such as methylatedcytosine residues in a nuclear gene of interest, such as FoxP3 or CD45,and/or a cytokine, lipolytic protein, lipogenic protein, adiponectinreceptor, or an mRNA molecule encoding one of these proteins, and/or anacetylated amino acid or methylated metabolite such as those describedherein).

In certain embodiments, the allergy is selected from the groupconsisting of food allergy, seasonal allergy, pet allergy, hives, hayfever, allergic conjunctivitis, poison ivy allergy oak allergy, moldallergy, drug allergy, dust allergy, cosmetic allergy, and chemicalallergy.

In some cases, the allograft rejection is selected from the groupconsisting of skin graft rejection, bone graft rejection, vasculartissue graft rejection, ligament graft rejection, and organ graftrejection.

The ligament graft rejection described above may be selected from thegroup consisting of cricothyroid ligament graft rejection, periodontalligament graft rejection, suspensory ligament of the lens graftrejection, palmar radiocarpal ligament graft rejection, dorsalradiocarpal ligament graft rejection, ulnar collateral ligament graftrejection, radial collateral ligament graft rejection, suspensoryligament of the breast graft rejection, anterior sacroiliac ligamentgraft rejection, posterior sacroiliac ligament graft rejection,sacrotuberous ligament graft rejection, sacrospinous ligament graftrejection, inferior pubic ligament graft rejection, superior pubicligament graft rejection, anterior cruciate ligament graft rejection,lateral collateral ligament graft rejection, posterior cruciate ligamentgraft rejection, medial collateral ligament graft rejection, cranialcruciate ligament graft rejection, caudal cruciate ligament graftrejection, and patellar ligament graft rejection.

The organ graft rejection may be selected from the group consisting ofheart graft rejection, lung graft rejection, kidney graft rejection,liver graft rejection, pancreas graft rejection, intestine graftrejection, and thymus graft rejection.

In some embodiments, the graft-versus-host disease may arise from a bonemarrow transplant or transplant of one or more blood cells selected fromthe group consisting of hematopoietic stem cells, common myeloidprogenitor cells, common lymphoid progenitor cells, megakaryocytes,monocytes, basophils, eosinophils, neutrophils, macrophages, T-cells,B-cells, natural killer cells, and dendritic cells.

In embodiments of the above methods of treatment, the method may furtherinclude administering to the subject an additional therapeutic agent. Insome cases, the additional therapeutic agent is selected from the groupconsisting of tumor necrosis factor-alpha (TNFα), a tumor necrosisfactor receptor 2 (TNFR2) agonist, an immunotherapy agent (e.g., ananti-CTLA-4 agent, an anti-PD-1 agent, an anti-PD-L1 agent, ananti-PD-L2 agent, a TNF-α cross-linking agent, a TRAIL cross-linkingagent, a CD27 agent, a CD30 agent, a CD40 agent, a 4-1 BB agent, a GITRagent, an OX40 agent, a TRAILR1 agent, a TRAILR2 agent, and a TWEAKRagent), and combinations thereof.

In embodiments of any of the above methods of treatment, the BCG may beadministered in a unit dosage form including between about 5×10⁵ andabout 1×10⁷ colony forming units (cfu) per 0.1 milligrams of BCG (e.g.,between about 1×10⁶ and about 6×10⁶ cfu per 0.1 milligrams of BCG, suchas between about 1.8×10⁶ and about 3.9×10⁶ cfu per 0.1 milligrams ofBCG) In some embodiments, administration of BCG modulates a methylationstate of one or more deoxyribonucleotides in the subject. For instance,the administering may promote methylation or demethylation of one ormore deoxyribonucleotides in the subject. The one or moredeoxyribonucleotides may be located within a gene that encodes atranscription factor (e.g., FoxP3) or a protein that is expressed on thesurface of a T-cell (e.g., CD45). In certain embodiments, the one ormore deoxyribonucleotides include cytosine residues.

In some cases, the administration of BCG promotes an increase in thelevel of one or more proteins in the subject and/or the level of one ormore mRNA molecules encoding these proteins. The proteins may include acytokine, such as interleukin-6 (IL-6), tumor necrosis factor (TNF), orinterferon-gamma (IFNγ). In other embodiments, the one or more proteinsinclude a lipolytic protein, such as acyl co-enzyme A oxidase, carnitinepalmitoyltransferase, lipase, or uncoupling protein.

In other embodiments, the administration of BCG may promote a decreasein the level of one or more proteins in the subject and/or the level ofone or more mRNA molecules encoding these proteins.

For instance, the one or more proteins may include a lipogenic protein,such as acetyl co-enzyme A carboxylase α, acetyl co-enzyme A carboxylaseβ, fatty acid synthase, glyceraldehydes-6-phosphate dehydrogenase,stearoyl-CoA saturase, malic enzyme, or glucose-6-phosphatedehydrogenase. In some embodiments, the one or more proteins include anadiponectin receptor, such as adiponectin receptor 1 or adiponectinreceptor 2.

In some embodiments, the administration of BCG promotes acetylation ofone or more amino acids in the subject, such as alanine, aspartic acid,serine, threonine, histidine, 3-methylhistidine, valine, lysine, ormethionine. Additionally or alternatively, the administration maypromote methylation of one or more metabolites in the subject, such asN-α-acetylhistidine, glutaconic acid, glutarylcarnitine, lysine, andcysteine. In some embodiments, the administration of BCG promotesdemethylation of one or more metabolites in the subject, such as4-methyl-2-oxopentanoic acid or 3-methyl-2-oxobutyric acid.

In some embodiments, the administration of BCG promotes a decrease inthe level of one or more lysine acetyltransferases (KATs, such as KAT2A,KAT2B, KAT5, KAT6A, KAT6B, KAT7, or KAT8), histone acetyltransferases,histone deacetylases (HDACs, such as HDAC1, HDAC2, HDAC3, HDAC4, HDAC5,HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, HDAC1P1, HDAC1P2, SIRT1,SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, or SIRT7), or one or more histones(e.g., a H2A, H2B, H3, or H4 family histone).

In embodiments of any of the above-described methods of treatment, thesubject may exhibit a reduction in total cholesterol of between about 1%and about 40% (e.g., between about 5% and about 40%) relative to acontrol subject not treated with the BCG (e.g., a control subject of thesame age, sex, and/or weight of the subject being treated). In somecases, the subject exhibits this reduction in total cholesterol withinabout 3 months to about 7 years after being treated with the BCG. Insome embodiments, the subject exhibits a reduction in LDL of betweenabout 5% and about 60%, e.g., within about 3 months to about 7 yearsafter being treated with the BCG. Additionally or alternatively, asubject may exhibit a reduction in glycated hemoglobin of between about5% and about 30%, e.g., within about 2 weeks to about 7 years afterbeing treated with the BCG. The reduction in cholesterol, LDLs, and/orglycated hemoglobin may be maintained, e.g., for between about 1 yearand about 8 years, or more.

In another aspect, the invention provides a method of diagnosing asubject as having a disease, the method including:

-   -   a) determining a quantity of methylated cytosine residues in a        sample of nuclear DNA isolated from the subject; and    -   b) comparing the quantity to a quantity of methylated cytosine        residues in a reference sample (e.g., a sample isolated from a        control subject not having the disease, such as a subject of the        same age, gender, and/or weight),

such that a determination that the quantity of methylated cytosineresidues in the sample of nuclear DNA isolated from the subject isgreater than or less than the quantity of methylated cytosine residuesin the reference sample indicates that the subject has the disease.

The method may further include determining whether the subject is likelyto respond to treatment with a therapeutic agent for the disease, suchthat a determination that the quantity of methylated cytosine residuesin the sample of nuclear DNA isolated from the subject is greater thanor less than the quantity of methylated cytosine residues in thereference sample indicates that the subject is likely to respond to thetreatment. In some cases, the quantity of methylated cytosine residuesin the sample of nuclear DNA isolated from the subject is greater thanthe quantity of methylated cytosine residues in the reference sample by1% or more (e.g., by 5%, 10%, or more). In other embodiments, thequantity of methylated cytosine residues in the sample of nuclear DNAisolated from the subject is less than the quantity of methylatedcytosine residues in the reference sample by 1% or more (e.g., by 5%,10%, or more). In these cases, the methylated cytosine residues may belocated within a gene that encodes a transcription factor (e.g., FoxP3)or a protein that is expressed on the surface of a T-cell (e.g., CD45).

In another aspect, the invention provides a method of determiningwhether a subject previously administered a therapeutic agent for thetreatment of a disease would benefit from receiving one or moreadditional doses of the therapeutic agent, the method including:

-   -   a) determining a quantity of methylated cytosine residues in a        sample of nuclear DNA isolated from the subject; and    -   b) comparing the quantity to a quantity of methylated cytosine        residues in a reference sample (e.g., a prior sample that has        been previously isolated from the subject),

such that a determination that the quantity of methylated cytosineresidues in the sample of nuclear DNA isolated from the subject iswithin 10% of the quantity of methylated cytosine residues in thereference sample indicates that the subject would benefit from one ormore additional doses of the therapeutic agent. In some cases, adetermination that the quantity of methylated cytosine residues in thesample of nuclear DNA isolated from the subject is within 5% of (e.g.,within 1% of or the same as) the quantity of methylated cytosineresidues in the reference sample indicates that the subject wouldbenefit from one or more additional doses of the therapeutic agent. Inthese cases, the methylated cytosine residues may be located within agene that encodes a transcription factor (e.g., FoxP3) or a protein thatis expressed on the surface of a T-cell (e.g., CD45). In someembodiments of the invention, the method includes isolating apolynucleotide including one or more cytosine residues and treating thepolynucleotide with bisulfite. Optionally, the polynucleotide is thenamplified using a polymerase chain reaction (PCR).

In an additional aspect, the invention provides a method of diagnosing asubject as having a disease, the method including:

-   -   a) i) determining a level of one or more substances selected        from the group consisting of an mRNA molecule encoding a        cytokine or lipolytic protein, a protein selected from the group        consisting of a cytokine and a lipolytic protein, an acetylated        amino acid selected from the group consisting of        N-acetylalanine, N-acetylaspartic acid, N-acetylserine,        N-acetylthreonine, N-acetylhistidine,        N-acetyl-3-methylhistidine, N-acetylvaline, and        N-α-acetyllysine, and N-acetylmethionine, or a methylated        metabolite selected from the group consisting of        N-α-acetyl-3-methylhistidine, 3-methylglutaconic acid,        3-methylglutarylcarnitine, and N-ε-trimethyllysine in a sample        from the subject; and        -   (ii) comparing the level of the one or more substances to            the level of the one or more substances in a reference            sample (e.g., a sample isolated from a control subject not            having the disease, such as a subject of the same age,            gender, and/or weight), such that a determination that the            level of the one or more substances in the sample from the            subject having the disease is less than the level of the one            or more substances in the reference sample indicates that            the subject has the disease; or    -   b) (i) determining a level of one or more substances selected        from the group consisting of an mRNA molecule encoding a        lipogenic protein, an adiponectin receptor, a lysine        acetyltransferase, a histone acetyltransferase, a histone        deacetylase, or a histone, a protein selected form the group        consisting of a lipogenic protein, an adiponectin receptor, a        lysine acetyltransferase, a histone acetyltransferase, a histone        deacetylase, and a histone, or a methylated metabolite selected        from the group consisting of 4-methyl-2-oxopentanoic acid and        3-methyl-2-oxobutyric acid in a sample from the subject; and        -   (ii) comparing the level of the one or more substances to            the level of the one or more substances in a reference            sample (e.g., a sample isolated from a control subject not            having the disease, such as a subject of the same age,            gender, and/or weight), such that a determination that the            level of the one or more substances in the sample from the            subject having the disease is greater than the level of the            one or more substances in the reference sample indicates            that the subject has the disease.

In some embodiments, the method further includes determining whether thesubject is likely to respond to treatment with a therapeutic agent forthe disease, such that a determination that the level of the one or moresubstances listed in (a) in the sample from the subject having thedisease is less than the level of the one or more substances in thereference sample indicates that the subject is likely to respond to thetreatment, or such that a determination that the level of the one ormore substances listed in (b) in the sample from the subject having thedisease is greater than the level of the one or more substances in thereference sample indicates that the subject is likely to respond to thetreatment.

In another aspect, the invention provides a method of determiningwhether a subject previously administered a therapeutic agent for thetreatment of a disease would benefit from receiving one or moreadditional doses of the therapeutic agent, the method including:

-   -   a) (i) determining a level of one or more substances selected        from the group consisting of a an mRNA molecule encoding a        cytokine or lipolytic protein, a protein selected from the group        consisting of a cytokine and a lipolytic protein, an acetylated        amino acid selected from the group consisting of        N-acetylalanine, N-acetylaspartic acid, N-acetylserine,        N-acetylthreonine, N-acetylhistidine,        N-acetyl-3-methylhistidine, N-acetylvaline, and        N-α-acetyllysine, and N-acetylmethionine, or a methylated        metabolite selected from the group consisting of        N-α-acetyl-3-methylhistidine, 3-methylglutaconic acid,        3-methylglutarylcarnitine, and N-ε-trimethyllysine in a sample        from the subject; and        -   (ii) comparing the level of the one or more substances to            the level of the one or more substances in a reference            sample (e.g., a prior sample that has been previously            isolated from the subject), such that a determination that            the level of the one or more substances in the sample from            the subject is the same as or less than the level of the one            or more substances in the reference sample indicates that            the subject would benefit from additional doses of the            therapeutic agent, or    -   b) (i) determining a level of one or more substances selected        from the group consisting of an mRNA molecule encoding a        lipogenic protein, an adiponectin receptor, a lysine        acetyltransferase, a histone acetyltransferase, a histone        deacetylase, or a histone, a protein selected form the group        consisting of a lipogenic protein, an adiponectin receptor, a        lysine acetyltransferase, a histone acetyltransferase, a histone        deacetylase, and a histone, or a methylated metabolite selected        from the group consisting of 4-methyl-2-oxopentanoic acid and        3-methyl-2-oxobutyric acid in a sample from the subject; and        -   (ii) comparing the level of the one or more substances to            the level of the one or more substances in a reference            sample (e.g., a prior sample that has been previously            isolated from the subject), such that a determination that            the level of the one or more substances in the sample from            the subject is the same as or greater than the level of the            one or more substances in the reference sample indicates            that the subject would benefit from additional doses of the            therapeutic agent.

In some cases, the method includes determining the level of a protein,such as a cytokine (e.g., IL-6, TNF, and IFNγ) or lipolytic protein(e.g., acyl co-enzyme A oxidase, carnitine palmitoyltransferase, lipase,and uncoupling protein), or an mRNA molecule encoding one of theseproteins. In other embodiments, the method includes determining thelevel of an acetylated amino acid selected from the group consisting ofN-acetylalanine, N-acetylaspartic acid, N-acetylserine,N-acetylthreonine, N-acetylhistidine, N-acetyl-3-methylhistidine,N-acetylvaline, and N-α-acetyllysine, and N-acetylmethionine. The methodmay include determining the level of a methylated metabolite selectedfrom the group consisting of N-α-acetyl-3-methylhistidine,3-methylglutaconic acid, 3-methylglutarylcarnitine, andN-ε-trimethyllysine. In some embodiments, the method includesdetermining the level of a protein, such as a lipogenic protein (e.g.,acetyl co-enzyme A carboxylase α, acetyl co-enzyme A carboxylase β,fatty acid synthase, glyceraldehydes-6-phosphate dehydrogenase,stearoyl-CoA saturase, malic enzyme, or glucose-6-phosphatedehydrogenase), an adiponectin receptor (e.g., adiponectin receptor 1 oradiponectin receptor 2), a lysine acetyltransferase (e.g., KAT2A, KAT2B,KAT5, KAT6A, KAT6B, KAT7, and KAT8), a histone acetyltransferase, ahistone deacetylase (e.g., HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6,HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, HDAC1P1, HDAC1P2, SIRT1, SIRT2,SIRT3, SIRT4, SIRT5, SIRT6, or SIRT7), or a histone (e.g., a H2A, H2B,H3, or H4 family histone). In some cases, the method includesdetermining the level of a protein selected from the group consisting ofa lipogenic protein, an adiponectin receptor, a lysineacetyltransferase, a histone acetyltransferase, a histone deacetylase,and a histone, or an mRNA molecule encoding one of these proteins. Insome embodiments, the method includes determining the level of amethylated metabolite selected from the group consisting of4-methyl-2-oxopentanoic acid and 3-methyl-2-oxobutyric acid.

In some cases, the level of the mRNA molecule is determined byperforming an assay selected from the group consisting of reversetranscription PCR (RT-PCR) and a Northern blot. In other embodiments,the level of the protein is determined by performing an assay selectedfrom the group consisting of an immunoblot and an enzyme-linkedimmunosorbant assay (ELISA). Additionally or alternatively, the level ofthe acetylated amino acid or the methylated metabolite is determined bynuclear magnetic resonance (NMR) spectroscopy.

In some embodiments of the above-described aspects of the invention, theprior sample has been isolated between about 24 hours and about 5 yearsbefore making the determination, such as about 1 month and about 1 yearprior to making the determination. In some cases, the prior sample wasisolated prior to the subject being administered the therapeutic agent.

In some embodiments of the above-described aspects of the invention, thedisease is a condition associated with elevated levels of cholesterol,such as hypercholesterolemia, hyperlipidemia, coronary heart disease,peripheral arterial disease (PAD), peripheral vascular disease,hypertension, stroke, diabetes, metabolic syndrome, obesity, and insulinresistance. In other cases, the disease is an autoimmune disease, aneurological condition, an allergy, allograft rejection,graft-versus-host disease, asthma, macular degeneration, muscularatrophy, a disease related to miscarriage, atherosclerosis, bone loss, amusculoskeletal disease, or obesity, e.g., as described herein.

In some embodiments of the above-described methods of the invention, thetherapeutic agent is BCG. The methods of the invention may furtherinclude administering an effective amount of BCG to the subject. The BCGmay be administered, e.g., in a unit dosage form including between about5×10⁵ and about 1×10⁷ colony forming units (cfu) per 0.1 milligrams ofBCG, such as between about 1×10⁶ and about 6×10⁶ cfu per 0.1 milligramsof BCG, preferably between about 1.8×10⁶ and about 3.9×10⁶ cfu per 0.1milligrams of BCG. The BCG may be administered to the subject about onceevery 1-20 years (e.g., between about once every 1-10 years, such asabout once every 5 years). The patient may be administered a total ofabout 1-20 doses of the BCG (e.g., a total of about 2-5 doses of theBCG, such as a total of 2 doses of BCG).

In other embodiments, the therapeutic agent is a hypolipidemic agentselected from the group consisting of a HMG-CoA reductase inhibitor,niacin, a fibric acid derivative, a cholesterol absorption inhibitor,and a lipolytic agent, e.g., as described herein.

In some embodiments of the invention, an additional therapeutic agent isadministered to the subject, such as TNFα, a TNFR2 agonist, or animmunotherapy agent (e.g., an anti-CTLA-4 agent, an anti-PD-1 agent, ananti-PD-L1 agent, an anti-PD-L2 agent, a TNF-α cross-linking agent, aTRAIL cross-linking agent, a CD27 agent, a CD30 agent, a CD40 agent, a4-1 BB agent, a GITR agent, an OX40 agent, a TRAILR1 agent, a TRAILR2agent, or a TWEAKR agent).

In embodiments of any of the above-described aspects of the invention,the subject is a mammal (e.g., a human).

In yet another aspect, the invention provides a kit including BCG and apackage insert instructing a user of the kit to treat a subjectaccording to any of the methods of the invention described herein. Thekit may further include an additional therapeutic agent, e.g., asdescribed above.

An additional aspect of the invention relates to a kit including anagent that can be used to determine the methylation state of one or morecytosine residues, such as a bisulfite salt, as well as a package insertinstructing a user of the kit to perform a method of the invention(e.g., to determine the quantity of methylated cytosine residues in agene of interest according to a method of the invention).

In still another aspect, the invention provides a kit including an agentthat can be used to detect one or more mRNA molecules (e.g., acomplementary polynucleotide capable of hybridizing with the mRNAmolecule of interest, e.g., by Watson-Crick base pairing), in additionto a package insert instructing a user of the kit to perform a method ofthe invention (e.g., to measure a level of one or more mRNA moleculesaccording to a method of the invention).

The invention additionally provides a kit that includes a package insertinstructing a user of the kit to perform a method of diagnosing asubject or assessing a subject for the benefit of administering one ormore additional doses of a therapeutic agent (e.g., BCG) as describedherein.

In another aspect, the invention features a method of reducing the levelof glucose (e.g., by 1% or more, and/or by at least 10 mg/dL, such as bybetween 10 mg/dL and 100 mg/dL or more, such as by 10 mg/dL, 20 mg/dL,30 mg/dL, 40 mg/dL, 50 mg/dL, 60 mg/dL, 70 mg/dL, 80 mg/dL, 90 mg/dL,100 mg/dL, or more) in the blood of a subject (e.g., a mammaliansubject, such as a human subject) in need thereof includingadministering BCG to the subject.

The subject may be a hyperglycemic subject, such as a subject having (i)a chronically, acutely, or persistently elevated blood glucose level,such as a chronic, acute, or persistent blood glucose level of over 100mg/dL (e.g., a subject having a chronic, acute, or persistent bloodglucose level of over 126 mg/dL, such as a subject having a chronic,acute, or persistent blood glucose level of 130 mg/dL, 140 mg/dL, 150mg/dL, 160 mg/dL, 170 mg/dL, 180 mg/dL, 190 mg/dL, 200 mg/dL, 210 mg/dL,220 mg/dL, 230 mg/dL, 240 mg/dL, 250 mg/dL, or more), (ii) a subjectsuffering from a disease associated with hyperglycemia, such as type 2diabetes, noninsulin-dependent diabetes mellitus (NIDDM), nonalcoholicsteatohepatitis (NASH), metabolic syndrome, cystic fibrosis, druginduced hyperglycemia, insulin resistance syndromes, diseases caused bygenetic mutations in the pancreas, cancer, infection, Leprechaunism,Rabson Mandenhall syndrome, lipoatrophic diabetes, pancreatitis, trauma,hemochromatoisis, fibrocalculous pancreatopathy, acromegaly, Cushingssyndrome, glucagonoma, pheochromocytoma, hyperthyroism, somatostatinoma,aldosteroma, infections associated with beta cell destruction, Rubella,coxsachie virus B, mumps, cytomegatolovirus infection, adenovirusinfection, a genetic syndrome, stiff person syndrome, anti-insulinreceptor abnormalities, liver disease, and renal failure, and/or (iii) asubject exhibiting an increase in blood glucose concentration relativeto a previous measurement of blood glucose in the blood of the subject,such as an increase of from about 10 mg/dL to about 200 mg/dL over thecourse of from about 1 week to about 5 years (e.g., an increase in bloodglucose level of from about 10 mg/dL, 20 mg/dL, 30 mg/dL, 40 mg/dL, 50mg/dL, 60 mg/dL, 70 mg/dL, 80 mg/dL, 90 mg/dL, 100 mg/dL, 110 mg/dL, 120mg/dL, 130 mg/dL, 140 mg/dL, 150 mg/dL, 160 mg/dL, 170 mg/dL, 180 mg/dL,190 mg/dL, 200 mg/dL, or more, over the course of 1 week, 1 month, 6months, 1 year, 2 years, 3 years, 4 years, 5 years, or longer).

In some embodiments, the administration of BCG lowers the level ofglucose in the blood of the subject by about 0.1%, 0.2%, 0.3%, 0.4%,0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, or more (e.g., byabout 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%,40%, 50%, or more). In some embodiments, the administration of BCGlowers the level of glucose in the blood of the subject by from about10% to about 40% (e.g., by about 10%, 20%, 30%, or 40%). Theadministration may lower the level of glucose in the blood of thesubject within about 1 week to about 8 years (e.g., within about 1 week,2 weeks, 3 weeks, 4 weeks, 6 months, 1 year, 2 years, 3 years, 4 years,5 years, 6 years, 7 years, 8 years, or more, such as from about 1 weekto about 8 years, from about 6 months to about 5 years, or from about 1year to about 3 years) after being treated with the BCG. In someembodiments, the blood glucose level of the subject is stabilizedfollowing administration of BCG, for instance, such that it does notincrease (e.g., by more than 15%, such as by not more than about 15%,14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, or less) for a period offrom about 1 week to about 8 years following said administration (e.g.,1 week, 2 weeks, 3 weeks, 4 weeks, 6 months, 1 year, 2 years, 3 years, 4years, 5 years, 6 years, 7 years, 8 years, or more). In someembodiments, the invention features methods of stabilizing the level ofglucose in the blood of a subject, for instance, by administration ofBCG as described herein. In some embodiments, the invention featuresmethods of preventing an increase in the level of glucose in the bloodof a subject, for instance, by administration of BCG as describedherein.

In some embodiments, the administration of BCG lowers the level ofglycated hemoglobin in the blood of the subject, for instance, by about5% or more (e.g., by about 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%,or more). In some embodiments, the administration of BCG lowers thelevel of glycated hemoglobin in the blood of the subject by about 15% ormore (e.g., by about 20%, 30%, 40%, 50%, or more). The administration ofBCG may lower the level of glycated hemoglobin in the blood of thesubject within about 2 weeks to about 8 years after being treated withthe BCG (e.g., within about 2 weeks, 3 weeks, 4 weeks, 6 months, 1 year,2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, or more).

Administration of BCG can cause an increase in the rate of glycolysis inthe subject, for instance, by increasing the expression of one or moreglycolytic enzymes in the subject. Administration of BCG can cause anincrease in flux through the pentose phosphate shunt in the subject. BCGadministration can cause a decrease in the rate of oxidativephosphorylation (e.g., of adenosine diphosphate) in the subject.

In some embodiments, administration of BCG increases the level oflactate and/or 1,5-anhydroglucitol in the blood of the subject. In someembodiments, administration of BCG lowers the level of α-ketobutyrateand/or 2-hydroxybutyrate in the blood of the subject.

In some embodiments, administration of BCG increases the expression ofhypoxia-inducible factor 1-α (HIF1-α) in the blood of the subject, forinstance, in a lymphocyte (e.g., a peripheral blood lymphocyte) or in amonocyte in the subject. Expression of HIF1-α may be assessed, forexample, by monitoring HIF1-α mRNA or protein expression in a sample(e.g., a blood sample) isolated from the subject following BCGadministration. In some embodiments, administration of BCG increasesexpression of HIF-1α mRNA by from about 3-fold to about 6-fold.

Administration of BCG to the subject may increase expression of aglucose transporter in the blood of the subject (e.g., relative to ameasurement of the glucose transporter in the blood of the subject priorto administration to BCG and/or relative to a measurement of the glucosetransporter in the blood of a healthy subject not suffering fromhyperglycemia or a disease associated therewith, such as a healthysubject of the same age and/or gender as the subject), such as solutecarrier family 2 member 6 (SLC2A6), for instance, by 1%, 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, or more. Expression of the glucosetransporter may be assessed, for example, by monitoring glucosetransporter mRNA or protein expression in a sample isolated from thesubject following BCG administration. In some embodiments,administration of BCG increases the expression of a glycolytic enzyme inthe blood of the subject, such as hexokinase 2 (HK2),glucose-6-phosphate isomerase (G6PI), triosephosphate isomerase 1(TPI1), galactokinase 1 (GALK1), and galactose mutarotase (GALM).Expression of the glycolytic enzyme may be assessed, for example, bymonitoring glucose transporter mRNA or protein expression in a sample(e.g., a blood sample) isolated from the subject following BCGadministration.

In some embodiments, administration of BCG to the subject reduces theexpression of an enzyme that promotes flux through the Krebs cycle inthe blood of the subject (e.g., relative to a measurement of the enzymein the blood of the subject prior to administration to BCG and/orrelative to a measurement of the enzyme in the blood of a healthysubject not suffering from hyperglycemia or a disease associatedtherewith, such as a healthy subject of the same age and/or gender asthe subject), such as adenosine triphosphate citrate lyase (ACLY),aconitase 2 (ACO2), citrate synthase (CS), dihydrolipoamidedehydrogenase (DLD), oxoglutarate dehydrogenase (OGDH), succinatedehydrogenase iron-sulfur complex subunit B (SDHB), and succinate-CoAligase subunit α (SUCLG1), for instance, by 1%, 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, or more. Expression of the enzyme that promotesflux through the Krebs cycle can be assessed, for instance, bymonitoring enzyme mRNA or protein expression in a sample isolated fromthe subject following BCG administration.

The administration of BCG may lower the level of one or more of theabove-referenced substances in the blood of the subject within about 2weeks to about 8 years after being treated with the BCG (e.g., withinabout 2 weeks, 3 weeks, 4 weeks, 6 months, 1 year, 2 years, 3 years, 4years, 5 years, 6 years, 7 years, 8 years, or more).

In some embodiments, the subject has a blood glucose level of about 100mg/dL or greater prior to administration of the BCG (e.g., 100 mg/dL,110 mg/dL, 120 mg/dL, 130 mg/dL, 140 mg/dL, 150 mg/dL, 160 mg/dL, 170mg/dL, 180 mg/dL, 190 mg/dL, 200 mg/dL, 210 mg/dL, 220 mg/dL, 230 mg/dL,240 mg/dL, 250 mg/dL, 260 mg/dL, 270 mg/dL, 280 mg/dL, 290 mg/dL, 300mg/dL or more, such as a blood glucose level of about 126 mg/dL orgreater (e.g., 130 mg/dL, 140 mg/dL, 150 mg/dL, 160 mg/dL, 170 mg/dL,180 mg/dL, 190 mg/dL, 200 mg/dL, 210 mg/dL, 220 mg/dL, 230 mg/dL, 240mg/dL, 250 mg/dL, 260 mg/dL, 270 mg/dL, 280 mg/dL, 290 mg/dL, 300 mg/dLor more). In some embodiments, the subject has a blood glucose level ofabout 200 mg/dL or more prior to administration of the BCG. The subjectmay have a blood glucose level that is higher than a blood glucose levelthat has previously been observed for the subject, such as prior toadministration of the BCG. In some embodiments, the subject haspreviously been observed as having a blood glucose level of less thanabout 200 mg/dL, such as less than about 126 mg/dL, such as a bloodglucose level of about 100 mg/dL or less, for instance, prior toadministration of the BCG.

In some embodiments, the subject is suffering from a disease associatedwith an elevated blood glucose level, such as type 2 diabetes,noninsulin-dependent diabetes mellitus (NIDDM), nonalcoholicsteatohepatitis (NASH), metabolic syndrome, cystic fibrosis, druginduced hyperglycemia, insulin resistance syndromes, diseases caused bygenetic mutations in the pancreas, cancer, infection, Leprechaunism,Rabson Mandenhall syndrome, lipoatrophic diabetes, pancreatitis, trauma,hemochromatoisis, fibrocalculous pancreatopathy, acromegaly, Cushingssyndrome, glucagonoma, pheochromocytoma, hyperthyroism, somatostatinoma,aldosteroma, infections associated with beta cell destruction, Rubella,coxsachie virus B, mumps, cytomegatolovirus infection, adenovirusinfection, a genetic syndrome, stiff person syndrome, anti-insulinreceptor abnormalities, liver disease, and renal failure. In someembodiments, the drug induced hyperglycemia is induced by one or moreagents selected from the group consisting of steroids, cortisol,thiazides, diazocide, calcineurin inhibitors, oral contraceptives, betaadrenergic agonists, nicotinic acid, pentamidine, alpha interferon,anti-psychotic agents, anti-retroviral agents, and rodenticides (e.g.,pyrinuron). In some embodiments, the cancer is pancreatic cancer. Insome embodiments, the genetic syndrome is selected from the groupconsisting of Down's syndrome, Klinefelter's syndrome, Turner syndrome,Woldfram syndrome, and Friendreich ataxia. In some embodiments, thesubject has undergone a pancreatectomy. The subject may exhibit one ormore mutations in a mitochondrial gene, such as hepatic nuclear factor 1(MODY3), glucokinase (MODY2), and hepatocyte nuclear factor 4-α (MODY1).

In some embodiments, BCG is administered to the subject in an amountsufficient to alleviate or reduce a symptom associated with the disease,such as polyphagia, polydipsia, polyuria, blurred vision, fatigue,cardiac arrhythmia, stupor, dry mouth, and poor wound healing.

In some embodiments, the BCG is administered in a unit dosage formcontaining between about 5×10⁵ and about 1×10⁷ cfu per 0.1 milligrams ofBCG, such as a unit dosage form containing between about 1×10⁶ and about6×10⁶ cfu per 0.1 milligrams of BCG (e.g., a unit dosage form containingbetween about 1.8×10⁶ and about 3.9×10⁶ cfu per 0.1 milligrams of BCG,such as 1.8×10⁶ cfu per 0.1 milligrams of BCG, 1.9×10⁶ cfu per 0.1milligrams of BCG, 2.0×10⁶ cfu per 0.1 milligrams of BCG, 2.1×10⁶ cfuper 0.1 milligrams of BCG, 2.2×10⁶ cfu per 0.1 milligrams of BCG,2.3×10⁶ cfu per 0.1 milligrams of BCG, 2.4×10⁶ cfu per 0.1 milligrams ofBCG, 2.5×10⁶ cfu per 0.1 milligrams of BCG, 2.6×10⁶ cfu per 0.1milligrams of BCG, 2.7×10⁶ cfu per 0.1 milligrams of BCG, 2.8×10⁶ cfuper 0.1 milligrams of BCG, 2.9×10⁶ cfu per 0.1 milligrams of BCG,3.0×10⁶ cfu per 0.1 milligrams of BCG, 3.1×10⁶ cfu per 0.1 milligrams ofBCG, 3.2×10⁶ cfu per 0.1 milligrams of BCG, 3.3×10⁶ cfu per 0.1milligrams of BCG, 3.4×10⁶ cfu per 0.1 milligrams of BCG, 3.5×10⁶ cfuper 0.1 milligrams of BCG, 3.6×10⁶ cfu per 0.1 milligrams of BCG,3.7×10⁶ cfu per 0.1 milligrams of BCG, 3.8×10⁶ cfu per 0.1 milligrams ofBCG, or 3.9×10⁶ cfu per 0.1 milligrams of BCG).

In some embodiments, BCG is administered to the subject by a route ofadministration described herein or known in the art. For instance, BCGmay be administered to the subject intradermally, subcutaneously, orpercutaneously (i.e., by an intradermal or subcutaneous route). In someembodiments, BCG is not administered to the subject intravenously.

In some embodiments, the BCG is administered to the subject about onceevery 1-20 years, such as about once every 1-10 years (e.g., about onceevery 5 years). In some embodiments, the subject is administered a totalof 1-20 doses of BCG, such as a total of 2-5 doses of BCG (e.g., a totalof 2 doses of BCG). The subject may be administered a first dose of BCGfollowed by a second dose of BCG from about 2 weeks to about 8 weeksafter administration of the first dose. For instance, the subject may beadministered a first dose of BCG followed by a second dose of BCG about4 weeks after administration of the first dose.

In some embodiments, subsequent doses of BCG (e.g., a second, third,fourth, or fifth, or greater) dose of BCG can be administered to thesubject, for instance, if the blood glucose concentration in the subjecthas increased by 10% or more (e.g., by about 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, or more) or by 20 mg/dL or more (e.g., by about 20mg/dL, 30 mg/dL, 40 mg/dL, 50 mg/dL, 60 mg/dL, 70 mg/dL, 80 mg/dL, 90mg/dL, 100 mg/dL, or more) over a period of from about 6 months to about5 years (e.g., 6 months, 1 year, 2 years, 3 years, 4 years, 5 years, ormore).

Additionally or alternatively, BCG administration can increase theexpression of nuclear receptor subfamily 1 group H member 3 (NR1H3) inthe subject (e.g., relative to a measurement of NR1H3 in the blood ofthe subject prior to administration to BCG and/or relative to ameasurement of NR1H3 in the blood of a healthy subject not sufferingfrom hyperglycemia or a disease associated therewith, such as a healthysubject of the same age and/or gender as the subject), for instance, by1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or more. NR1H3expression can be assessed, for instance, by monitoring NR1H3 mRNA orprotein expression. BCG administration can increase the expression ofcholesterol-suppressing genes, such as adenosine triphosphate bindingcassette subfamily A member 1 (ABCA1), adenosine triphosphate bindingcassette subfamily G (ABCG), apolipoprotein E(APOE), Fas cell surfacedeath receptor (FAS), and stearoyl-CoA desaturase (SCD1). Additionallyor alternatively, BCG administration can reduce the expression ofglucose-elevating genes, such as fructose-bisphosphatase 1 (FBP1),glucose-6-phosphate dehydrogenase (G6PD), and muscle pyruvate kinase(PKM).

In another aspect, the invention features a method of diagnosing asubject as having hyperglycemia or a disease associated therewith, themethod comprising:

-   -   a) i) determining a level of one or more substances selected        from the group consisting of glucose, cholesterol, LDL, a        triglyceride, glycated hemoglobin, an mRNA molecule encoding a        protein selected from the group consisting of FBP1, G6PD, and        PKM, a protein selected from the group consisting of FBP1, G6PD,        and PKM, an mRNA molecule encoding an enzyme that promotes flux        through the Krebs cycle, an enzyme that promotes flux through        the Krebs cycle, α-ketobutyrate, and 2-hydroxybutyrate in a        sample from the subject; and        -   (ii) comparing the level of the one or more substances to            the level of the one or more substances in a reference            sample, wherein a determination that the level of the one or            more substances in the sample from the subject is greater            than the level of the one or more substances in the            reference sample indicates that the subject has            hyperglycemia or disease associated with hyperglycemia; or    -   b) (i) determining a level of one or more substances selected        from the group consisting of an mRNA molecule encoding a        glycolytic enzyme, a glycolytic enzyme, an mRNA molecule        encoding a glucose transporter, a glucose transporter, an mRNA        molecule encoding HIF1-α, HIF1-α, an mRNA molecule encoding        NR1H3, NR1H3, lactate, and 1,5-anhydroglucitol in a sample from        the subject; and        -   (ii) comparing the level of the one or more substances to            the level of the one or more substances in a reference            sample, wherein a determination that the level of the one or            more substances in the sample from the subject is less than            the level of the one or more substances in the reference            sample indicates that the subject has hyperglycemia or            disease associated with hyperglycemia.

In some embodiments, the method further comprising determining whetherthe subject is likely to respond to treatment with a therapeutic agentfor the disease (e.g., BCG), wherein a determination that the level ofthe one or more substances listed in (a) in the sample from the subjecthaving the hyperglycemia or disease associated therewith is greater thanthe level of the one or more substances in the reference sampleindicates that the subject is likely to respond to the treatment, orwherein a determination that the level of the one or more substanceslisted in (b) in the sample from the subject having the disease is lessthan the level of the one or more substances in the reference sampleindicates that the subject is likely to respond to the treatment.

In some embodiments, the reference sample is a sample isolated from acontrol subject not having the disease. In some embodiments, the controlsubject is a subject of the same age and/or gender of the subject havingthe disease.

In some embodiments, the reference sample is a prior sample that hasbeen previously isolated from the subject. In some embodiments, theprior sample was isolated between about 24 hours and about 5 yearsbefore making the determination, such as between about 1 month and about1 year prior to making the determination. In some embodiments, the priorsample was isolated prior to the subject being administered thetherapeutic agent.

In another aspect, the invention features a method of determiningwhether a subject previously administered a therapeutic agent for thetreatment of a disease would benefit from receiving one or moreadditional doses of the therapeutic agent, the method comprising:

-   -   a) (i) determining a level of one or more substances selected        from the group consisting of glucose, cholesterol, LDL, a        triglyceride, glycated hemoglobin, an mRNA molecule encoding a        protein selected from the group consisting of FBP1, G6PD, and        PKM, a protein selected from the group consisting of FBP1, G6PD,        and PKM, an mRNA molecule encoding an enzyme that promotes flux        through the Krebs cycle, an enzyme that promotes flux through        the Krebs cycle, α-ketobutyrate, and 2-hydroxybutyrate in a        sample from the subject; and        -   (ii) comparing the level of the one or more substances to            the level of the one or more substances in a reference            sample, wherein a determination that the level of the one or            more substances in the sample from the subject is the same            as or greater than the level of the one or more substances            in the reference sample indicates that the subject would            benefit from one or more additional doses of the therapeutic            agent, or    -   b) (i) determining a level of one or more substances selected        from the group consisting of an mRNA molecule encoding a        glycolytic enzyme, a glycolytic enzyme, an mRNA molecule        encoding a glucose transporter, a glucose transporter, an mRNA        molecule encoding HIF1-α, HIF1-α, an mRNA molecule encoding        NR1H3, NR1H3, lactate, and 1,5-anhydroglucitol in a sample from        the subject; and        -   (ii) comparing the level of the one or more substances to            the level of the one or more substances in a reference            sample, wherein a determination that the level of the one or            more substances in the sample from the subject is the same            as or less than the level of the one or more substances in            the reference sample indicates that the subject would            benefit from one or more additional doses of the therapeutic            agent.

In some embodiments, the reference sample is a sample isolated from acontrol subject not having the disease. In some embodiments, the controlsubject is a subject of the same age and/or gender of the subject havingthe disease.

In some embodiments, the reference sample is a prior sample that hasbeen previously isolated from the subject. In some embodiments, theprior sample was isolated between about 24 hours and about 5 yearsbefore making the determination, such as between about 1 month and about1 year prior to making the determination. In some embodiments, the priorsample was isolated prior to the subject being administered thetherapeutic agent.

In some embodiments, the enzyme that promotes flux through the Krebscycle is selected from the group consisting of ACLY, ACO2, CS, DLD,OGDH, SDHB, and SUCLG1. In some embodiments, the glycolytic enzyme isselected from the group consisting of HK2, G6PI, TPI1, GALK1, and GALM.In some embodiments, the glucose transporter is SLC2A6.

In some embodiments, the level of the mRNA molecule is determined byperforming an assay selected from the group consisting of reversetranscription PCR (RT-PCR) and a Northern blot. In some embodiments, thelevel of the enzyme that promotes flux through the Krebs cycle,glycolytic enzyme, glucose transporter, HIF-1α, NR1H3, FBP1, G6PD, orPKM is determined by performing an assay selected from the groupconsisting of an immunoblot and an enzyme-linked immunosorbant assay(ELISA). In some embodiments, the level of the α-ketobutyrate,2-hydroxybutyrate, lactate, or 1,5-anhydroglucitol is determined bynuclear magnetic resonance (NMR) spectroscopy.

In some embodiments, the disease associated with hyperglycemia isselected from the group consisting of type 2 diabetes,noninsulin-dependent diabetes mellitus (NIDDM), nonalcoholicsteatohepatitis (NASH), metabolic syndrome, cystic fibrosis, druginduced hyperglycemia, insulin resistance syndromes, diseases caused bygenetic mutations in the pancreas, cancer, infection, Leprechaunism,Rabson Mandenhall syndrome, lipoatrophic diabetes, pancreatitis, trauma,hemochromatoisis, fibrocalculous pancreatopathy, acromegaly, Cushingssyndrome, glucagonoma, pheochromocytoma, hyperthyroism, somatostatinoma,aldosteroma, infections associated with beta cell destruction, Rubella,coxsachie virus B, mumps, cytomegatolovirus infection, adenovirusinfection, a genetic syndrome, stiff person syndrome, anti-insulinreceptor abnormalities, liver disease, and renal failure.

In some embodiments, the method comprising administering the therapeuticagent to the subject identified as a subject that would benefit from oneor more additional doses of the therapeutic agent (e.g., BCG). In someembodiments, the method comprises administering BCG to the subjectidentified as having hyperglycemia or a disease associated therewith. Insome embodiments, BCG is the only therapeutic agent administered to thesubject (e.g., the sole therapeutic agent).

In some embodiments, the subject is not administered an agent thatpromotes the expression of IL-2. In some embodiments, the subject is notadministered lymphotoxin or Lentinan. In some embodiments, the subjectis not administered TNF-α, a TNF-α agonist antibody, or an agent thatpromotes the expression of TNF-α other than BCG.

In another embodiment of the methods of treating and/or diagnosinghyperglycemia or conditions related to hyperglycemia, the patient to betreated does not have type 1 diabetes.

In another aspect, the invention features a method of inducing anincrease in the rate of aerobic glycolysis in a mammalian subject (e.g.,a human), the method comprising administering to the subject BCG. Therate of aerobic glycolysis in the subject may be measured, for instance,by measuring the expression of one or more glycolytic enzymes, such asan early glycolytic enzyme described herein, or by measuring the levelof a product of glycolysis or an associated fermentation pathway (e.g.,pyruvate or lactate) in the blood of the subject. In some embodiments,the rate of aerobic glycolysis is increased relative to a measurement ofaerobic glycolysis in the subject prior to administration of the BCG.The rate of aerobic glycolysis may be increased, for instance, by about0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%,5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, or more, e.g., as assessedby measuring the increase in the expression of one or more earlyglycolytic enzymes or by measuring the increase in the level of one ormore products of glycolysis or an associated fermentation pathway, suchas pyruvate or lactate, or by measuring the increase in the level ofHIF-1a or NR1H3 in the subject. In some embodiments, the administeringreduces the rate of oxidative phosphorylation of adenosine diphosphatein the subject relative to a measurement of oxidative phosphorylation ofadenosine diphosphate in the subject prior to administration of the BCG.The rate of oxidative phosphorylation may be decreased, for instance, byabout 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%,4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, or more, e.g., asassessed by measuring the decrease in the expression of one or moreearly enzymes that promote flux through the Krebs cycle or by measuringthe change in the level of one or more metabolites involved in the Krebscycle, such as a Krebs cycle metabolite described herein or known in theart.

In some embodiments, the administering reduces the level of one or moresubstances selected from the group consisting of glucose, cholesterol,LDL, a triglyceride, glycated hemoglobin, an mRNA molecule encoding aprotein selected from the group consisting of FBP1, G6PD, and PKM, aprotein selected from the group consisting of FBP1, G6PD, and PKM, anmRNA molecule encoding an enzyme that promotes flux through the Krebscycle, an enzyme that promotes flux through the Krebs cycle,α-ketobutyrate, and 2-hydroxybutyrate in the subject relative to ameasure of the substance in the subject prior to administration of theBCG.

In some embodiments, the administering increases the level of one ormore substances selected from the group consisting of an mRNA moleculeencoding a glycolytic enzyme, a glycolytic enzyme, an mRNA moleculeencoding a glucose transporter, a glucose transporter, an mRNA moleculeencoding HIF1-α, HIF1-α, an mRNA molecule encoding NR1H3, NR1H3,lactate, and 1,5-anhydroglucitol in the subject relative to a measure ofthe substance in the subject prior to administration of the BCG.

In some embodiments, the enzyme that promotes flux through the Krebscycle is selected from the group consisting of ACLY, ACO2, CS, DLD,OGDH, SDHB, and SUCLG1.

In some embodiments, the glycolytic enzyme is selected from the groupconsisting of HK2, G6PI, TPI1, GALK1, and GALM.

In some embodiments, the glucose transporter is SLC2A6.

Definitions

As used herein, the term “about” refers to a value that is within 10%above or below the value being described.

As used herein, the term “agonist” refers to a compound capable ofpromoting the activation of a receptor so as to potentiate a downstreamsignal transduction pathway. For instance, the terms “tumor necrosisfactor receptor 2 agonist” and “TNFR2 agonist” as used herein includecompounds that specifically bind TNFR2 in such a way that induces theactivation of this receptor which, in turn, promotes TRAF2/3- and/orNFκB-mediated cell proliferation. Agonists of TNFR2 include endogenousligands that activate the receptor, such as TNFα, which is capable ofbinding TNFR2 and inducing a conformational change that propagatessignal transduction events that lead to cell proliferation.

As used herein, “BCG” refers to Bacillus Calmette-Guerin, which is apreparation of Mycobacterium bovis, an attenuated strain ofMycobacterium turberculosis that is not virulent in humans. Examples ofBCG include a variety of substrains that have been developed by geneticmanipulation, including, e.g., the Pasteur, Phipps, Frappier, Mexico,Birkhaug, Sweden, Moreau, Japan-Tokyo, Copenhagen, TICE, Sanofi,Connaught, RIVM, Evans, MMC, and Glaxo substrains of BCG, among others,as well as genetic variants of these substrains. BCG substrains, as wellas the genetic differences between these substrains, are known in theart and are described, e.g., in Castillo-Rodal, et al., Infect Immun.74(3):1718-1724 (2006); as well as in Zhang, et al., Tubercle and LungDisease 76(1):43-50 (1995); the disclosures of each of which areincorporated herein by reference.

As used herein, a “colony forming unit” (cfu) refers to at least onecell that is capable of giving rise to a population of geneticallyidentical cells by mitotic cell proliferation. Colony forming unitsinclude a single cell, but may also be an aggregation of cells, such asa colony.

As used herein, the term “endogenous” describes a molecule (e.g., apolypeptide, nucleic acid, or cofactor) that is found naturally in aparticular organism (e.g., a human) or in a particular location withinan organism (e.g., an organ, a tissue, or a cell, such as a human cell).

As used herein, the terms “elevated blood glucose” and “hyperglycemic”are used interchangeably to characterize a subject having abnormallyhigh blood glucose concentrations and/or that may benefit from therapydescribed herein. Hyperglycemic subjects include those that exhibit oneor more, or all three, of the following characteristics: (i) a chronic,acute, or persistent blood glucose level of over 100 mg/dL (e.g., achronic, acute, or persistent blood glucose level of over 126 mg/dL,such as a chronic, acute, or persistent blood glucose level of 130mg/dL, 140 mg/dL, 150 mg/dL, 160 mg/dL, 170 mg/dL, 180 mg/dL, 190 mg/dL,200 mg/dL, 210 mg/dL, 220 mg/dL, 230 mg/dL, 240 mg/dL, 250 mg/dL, ormore), (ii) presenting with a disease associated with hyperglycemia,such as type 2 diabetes, noninsulin-dependent diabetes mellitus (NIDDM),nonalcoholic steatohepatitis (NASH), metabolic syndrome, cysticfibrosis, drug induced hyperglycemia, insulin resistance syndromes,diseases caused by genetic mutations in the pancreas, cancer, infection,Leprechaunism, Rabson Mandenhall syndrome, lipoatrophic diabetes,pancreatitis, trauma, hemochromatoisis, fibrocalculous pancreatopathy,acromegaly, Cushings syndrome, glucagonoma, pheochromocytoma,hyperthyroism, somatostatinoma, aldosteroma, infections associated withbeta cell destruction, Rubella, coxsachie virus B, mumps,cytomegatolovirus infection, adenovirus infection, a genetic syndrome,stiff person syndrome, anti-insulin receptor abnormalities, liverdisease, and renal failure, and/or (iii) an increase in blood glucoseconcentration relative to a previous measurement of blood glucose in theblood of the subject, such as an increase of from about 10 mg/dL toabout 200 mg/dL over the course of from about 1 week to about 5 years(e.g., an increase in blood glucose level of from about 10 mg/dL, 20mg/dL, 30 mg/dL, 40 mg/dL, 50 mg/dL, 60 mg/dL, 70 mg/dL, 80 mg/dL, 90mg/dL, 100 mg/dL, 110 mg/dL, 120 mg/dL, 130 mg/dL, 140 mg/dL, 150 mg/dL,160 mg/dL, 170 mg/dL, 180 mg/dL, 190 mg/dL, 200 mg/dL, or more, over thecourse of 1 week, 1 month, 6 months, 1 year, 2 years, 3 years, 4 years,5 years, or longer).

As used herein, the term “exogenous” describes a molecule (e.g., apolypeptide, nucleic acid, or cofactor) that is not found naturally in aparticular organism (e.g., a human) or in a particular location withinan organism (e.g., an organ, a tissue, or a cell, such as a human cell).Exogenous materials include those that are provided from an externalsource to an organism or to cultured matter extracted therefrom.

As used herein, the term “fusion protein” refers to a protein that isjoined via a covalent bond to another molecule. A fusion protein can bechemically synthesized by, e.g., an amide-bond forming reaction betweenthe N-terminus of one protein to the C-terminus of another protein.Alternatively, a fusion protein containing one protein covalently boundto another protein can be expressed recombinantly in a cell (e.g., aeukaryotic cell or prokaryotic cell) by expression of a polynucleotideencoding the fusion protein, for example, from a vector or the genome ofthe cell. A fusion protein may contain one protein that is covalentlybound to a linker, which in turn is covalently bound to anothermolecule. Examples of linkers that can be used for the formation of afusion protein include peptide-containing linkers, such as those thatcontain naturally occurring or non-naturally occurring amino acids. Incertain cases, it may be desirable to include D-amino acids in thelinker, as these residues are not present in naturally-occurringproteins and are thus more resistant to degradation by endogenousproteases. Linkers can be prepared using a variety of strategies thatare well known in the art, and depending on the reactive components ofthe linker, can be cleaved by enzymatic hydrolysis, photolysis,hydrolysis under acidic conditions, hydrolysis under basic conditions,oxidation, disulfide reduction, nucleophilic cleavage, or organometalliccleavage (Leriche et al., Bioorg. Med. Chem., 20:571-582, 2012).

As used herein, the term “percent (%) sequence identity” refers to thepercentage of amino acid (or nucleic acid) residues of a candidatesequence that are identical to the amino acid (or nucleic acid) residuesof a reference sequence after aligning the sequences and introducinggaps, if necessary, to achieve the maximum percent sequence identity(e.g., gaps can be introduced in one or both of the candidate andreference sequences for optimal alignment and non-homologous sequencescan be disregarded for comparison purposes). Alignment for purposes ofdetermining percent sequence identity can be achieved in various waysthat are within the skill in the art, for instance, using publiclyavailable computer software, such as BLAST, ALIGN, or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor measuring alignment, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For example, a reference sequence aligned for comparison with acandidate sequence may show that the candidate sequence exhibits from50% to 100% sequence identity across the full length of the candidatesequence or a selected portion of contiguous amino acid (or nucleicacid) residues of the candidate sequence. The length of the candidatesequence aligned for comparison purposes may be, for example, at least30%, (e.g., 30%, 40, 50%, 60%, 70%, 80%, 90%, or 100%) of the length ofthe reference sequence. When a position in the candidate sequence isoccupied by the same amino acid residue as the corresponding position inthe reference sequence, then the molecules are identical at thatposition.

As used herein, the term “operatively linked” in the context of apolynucleotide fragment is intended to mean that the two polynucleotidefragments are joined such that the amino acid sequences encoded by thetwo polynucleotide fragments remain in-frame.

As used herein, the term “regulatory sequence” includes promoters,enhancers and other expression control elements (e.g., polyadenylationsignals) that control the transcription or translation of the antibodychain genes. Such regulatory sequences are described, for example, inGoeddel, Gene Expression Technology: Methods in Enzymology 185 (AcademicPress, San Diego, Calif., 1990); incorporated herein by reference.

As used herein, the term “reference sample” refers to a measurement ofthe quantity of one or more substances, such as cholesterol, glucose,methylated or demethylated cytosine residues in one or more DNAmolecules, or the quantity of one or more mRNA molecules, proteins,acetylated amino acids, and/or methylated metabolites, that can becompared with a measurement of the same substance in a sample isolatedfrom a subject, e.g., in order to assess the likelihood of the subjectto respond to a particular therapy (such as BCG therapy) and/or todetermine if the subject would benefit from one or more subsequent dosesof a therapeutic agent after the subject has already received at leastone initial dose of a medicament to treat a disease or condition in thesubject. In the context of a method of determining whether a diseasethat the subject has been diagnosed as having is likely to be treatedwith a certain therapeutic regimen (e.g., administration of BCG), thereference sample may be a sample isolated from a healthy subject notsuffering from the disease being assessed, optionally of the same age,sex, and/or weight as the subject suffering from the disease. Thereference sample may alternatively be an accepted measurement of one ormore substances (e.g., the quantity of cholesterol, glucose, methylatedor demethylated cytosine residues in a DNA molecule, and/or the quantityof one or more mRNA molecules, proteins, acetylated amino acids, and/ormethylated metabolites) that is indicative of a normal physiologicalstate. In the context of a method of determining whether a patientsuffering from a certain disease or condition and that has already beenadministered at least one therapeutic agent (BCG), e.g., for thetreatment of the disease, would benefit from one or more additionaldoses of a medicament, the reference sample may be a sample previouslyisolated from the subject, such as a sample that was isolated from thesubject prior to earlier administration of a therapeutic agent. Thereference sample may alternatively be a measurement of one or moresubstances (e.g., the quantity of cholesterol, glucose, methylated ordemethylated cytosine residues in a DNA molecule, and/or the quantity ofone or more mRNA molecules, proteins, acetylated amino acids, and/ormethylated metabolites) that is indicative of an abnormal physiologicalstate, such as a physiological state that is characteristic of aparticular disease.

As used herein, the term “sample” refers to a specimen (e.g., blood,blood component (e.g., serum or plasma), urine, saliva, amniotic fluid,cerebrospinal fluid, tissue (e.g., placental or dermal), pancreaticfluid, chorionic villus sample, and cells) taken from a subject.Preferably, the sample is blood, a blood component (e.g., serum orplasma), or urine.

As used herein, the phrase “specifically binds” refers to a bindingreaction which is determinative of the presence of a receptor in aheterogeneous population of proteins and other biological molecules thatis recognized, e.g., by a ligand with particularity. A ligand thatspecifically binds to a receptor will bind to the receptor with a K_(D)of less than 100 nM. For example, a ligand that specifically binds to areceptor will bind to the receptor with a K_(D) of up to 100 nM (e.g.,between 1 pM and 100 nM). A ligand that does not exhibit specificbinding to a receptor or a domain thereof will exhibit a K_(D) ofgreater than 100 nM (e.g., greater than 500 nm, 1 μM, 100 μM, 500 μM, or1 mM) for that particular receptor or domain thereof. A variety of assayformats may be used to select ligands that specifically bind to aparticular receptor. For example, solid-phase ELISA assays are routinelyused to select ligands that specifically bind a receptor. See, Harlow &Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Press, NewYork (1988) and Harlow & Lane, Using Antibodies, A Laboratory Manual,Cold Spring Harbor Press, New York (1999), for a description of assayformats and conditions that can be used to determine specific proteinbinding.

As used herein, the terms “subject” and “patient” are interchangeableand refer to an organism that receives treatment for a particulardisease or condition as described herein (such as a condition associatedwith elevated levels of serum cholesterol or blood glucose) or that isdiagnosed as having a disease or condition according to the methodsdescribed herein. Examples of subjects and patients include mammals,such as humans, primates, pigs, goats, rabbits, hamsters, cats, dogs,guinea pigs, members of the bovidae family (such as cattle, bison,buffalo, and yaks, among others), cows, sheep, horses, and bison, amongothers, receiving treatment for diseases or conditions, for example,elevated cholesterol, LDLs, triglycerides, or blood glucose.

As used herein, the term “transfection” refers to any of a wide varietyof techniques commonly used for the introduction of exogenous DNA into aprokaryotic or eukaryotic host cell, e.g., electroporation, lipofection,calcium-phosphate precipitation, DEAE—dextran transfection and the like.

As used herein, the terms “treat” or “treatment” refer to therapeutictreatment, in which the object is to prevent or slow down (lessen) anundesired physiological change or disorder, such as the progression of adisease associated with an elevated level of cholesterol, such as heartdisease, a disease associated with an elevated level of blood glucose,such as type-2 diabetes, or an autoimmune disease. Beneficial or desiredclinical results include, but are not limited to, alleviation ofsymptoms, diminishment of extent of disease, stabilized (i.e., notworsening) state of disease, delay or slowing of disease progression,amelioration or palliation of the disease state, and remission (whetherpartial or total), whether detectable or undetectable. Those in need oftreatment include those already with the condition or disorder, as wellas those prone to have the condition or disorder or those in which thecondition or disorder is to be prevented.

As used herein, the term “vector” includes a nucleic acid vector, e.g.,a DNA vector, such as a plasmid, a RNA vector, virus or other suitablereplicon (e.g., viral vector). A variety of vectors have been developedfor the delivery of polynucleotides encoding exogenous proteins into aprokaryotic or eukaryotic cell. Examples of such expression vectors aredisclosed in, e.g., WO 1994/11026; incorporated herein by reference.Expression vectors of the invention contain a polynucleotide sequence aswell as, e.g., additional sequence elements used for the expression ofproteins and/or the integration of these polynucleotide sequences intothe genome of a mammalian cell. Certain vectors that can be used for therecombinant expression of proteins include plasmids that containregulatory sequences, such as promoter and enhancer regions, whichdirect gene transcription. Other useful vectors for recombinant proteinexpression contain polynucleotide sequences that enhance the rate oftranslation of these genes or improve the stability or nuclear export ofthe mRNA that results from gene transcription. These sequence elementsinclude, e.g., 5′ and 3′ untranslated regions, an internal ribosomalentry site (IRES), and polyadenylation signal site in order to directefficient transcription of the gene carried on the expression vector.The expression vectors of the invention may also contain apolynucleotide encoding a marker for selection of cells that containsuch a vector. Examples of a suitable marker include genes that encoderesistance to antibiotics, such as ampicillin, chloramphenicol,kanamycin, or nourseothricin.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph showing the effect of BCG on serum cholesterol levelsover the course of a multi-year study. The chart plots the percentchange in total cholesterol level as a function of time, in years,following administration of either BCG or a placebo (control) to a groupof patients presenting with high cholesterol levels. The datademonstrate that patients that were administered a placebo exhibited anaverage increase in total cholesterol of between about 10% and about 40%over the duration of the study, while patients that received BCG did notgenerally exhibit an increase in total serum cholesterol for up to sevenyears following the initial administration. These results support thefinding that BCG can be used according to the methods of the inventionto reduce or maintain serum cholesterol levels in patients (e.g.,patients diagnosed as having elevated cholesterol).

FIG. 2 is a graph showing the effect of BCG treatment on serum LDLconcentrations over the course of a multi-year study. The chart plotsthe percent change in LDL level as a function of time, in years,following administration of either BCG or a placebo (control) to a groupof patients presenting with high cholesterol levels. The datademonstrate the ability of BCG to promote a sustained reduction in LDLlevels, as patients that were administered BCG generally exhibited anincrease in LDL levels of 10% or less over the duration of the study,while patients that were administered a placebo typically exhibited anincrease in LDL levels of between 10% and 60%.

FIG. 3 is a graph showing the ability of BCG to induce a reduction inaverage glycated hemoglobin (HbA1c) levels in patients presenting withhigh serum cholesterol over the course of a multi-year investigation.The chart plots the fold change in HbA1c as a function of time, inyears, following administration of either BCG or a placebo (control) toa group of patients presenting with high serum cholesterol. The readout,HbA1c level, is an indicator of total blood glucose concentration andcan therefore be used to detect patients at risk of developing type Idiabetes. The data demonstrate that patients administered BCG exhibitedan average decrease in HbA1c levels of between 10% and 30% over thecourse of the study. Moreover, this reduction was generally sustainedover the eight-year measurement period. Taken together, the dataprovided in FIGS. 1-3 demonstrate the ability of BCG to lowercholesterol and HDL levels and to promote a long-term reduction in bloodglucose concentrations.

FIG. 4A is a table showing the average levels of various N-acetylatedamino acids in a group of patients following administration with eitherBCG (“Avg(BCGpost)”) or placebo (“Avg(Placebopost)”), as well as thep-value associated with the difference between these levels (“Ttest”).The data are shown as scaled levels such that all values are between 0and 2.5. FIG. 4B is a graph showing the changes in N-acetylated aminoacid concentrations listed in FIG. 4A. From left to right, vertical barsfor each N-acetylated amino acid appear in the order of average amongcontrol subjects (Avg(CTRL)), average among type-1 diabetes subjectssuffering from early or late onset of the disease (AVG(T1Dearly+late)),average following BCG treatment of type-1 diabetes subjects(AVG(BCGpost)), and average among placebo-treated subjects followingadministration of the placebo (Avg(Placebopost)). Taken together, thesedata demonstrate the ability of BCG to restore healthy levels ofN-acetylated amino acids in patients with type I diabetes as opposed topatients treated with a placebo. As shown in each figure, BCG is capableof promoting an increase in the concentrations of N-acetylalanine,N-acetylaspartate, N-acetylserine, N-acetylthreonine,N-acetyl-3-methylhistidine, N-acetylhistidine, N-acetylvaline,Na-acetyllysine, and N-acetylmethionine, which are depleted in patientssuffering from type I diabetes. BCG induces a decrease in theconcentrations of N-acetylphenylalanine, N-acetyltryptophan, andN-acetylarginine, which are elevated in patients with type I diabetes.Therefore, the changes promoted by BCG serve to restore N-acetylatedamino acid concentrations to healthy levels in patients suffering fromdisease associated with lipid and/or cholesterol metabolism, such astype I diabetes.

FIG. 5 is a graph showing the ability of BCG to restore healthy levelsof methylated metabolites in patients with type I diabetes relative topatients treated with a placebo. The chart shows the average levels ofvarious N-methylated metabolites amino acids in a group of patientsfollowing administration with either BCG (“Avg(BCGpost)”) or placebo(“Avg(Placebopost)”) relative to patients before receiving treatment ofany kind (“AVG(T1 Dearly+late)”) and relative to control patients notsuffering from type I diabetes (“Avg(CTRL)”). From left to right,vertical bars for each N-acetylated amino acid appear in the order ofaverage among control subjects (Avg(CTRL)), average among type-1diabetes subjects suffering from early or late onset of the disease(AVG(T1 Dearly+late)), average following BCG treatment of type-1diabetes subjects (AVG(BCGpost)), and average among placebo-treatedsubjects following administration of the placebo (Avg(Placebopost)). Thedata are shown as scaled levels such that all values are between 0 and2.5. The data demonstrate that BCG is capable of promoting an increasein the concentrations of N-acetyl-3-methylhistidine,3-methylglutaconate, 3-methylglutarylcarnitine, and Nε-trimethyllysine,which are depleted in patients suffering from type I diabetes. BCGinduces a decrease in the concentrations of 4-methyl-2-oxopentanoate and3-methyl-2-oxobutyrate, which are elevated in patients with type Idiabetes. Therefore, the changes promoted by BCG serve to restoreN-methylated metabolite concentrations to healthy levels.

FIG. 6 is a graph showing the effect of BCG treatment on the level ofvarious lipogenic and lipolytic enzymes in patients with type Idiabetes. The data demonstrate that BCG promotes an increase in theconcentrations of cytokines, such as IL-6, TNFα, and interferon γ(IFNγ), as well as in the levels of lipolytic factors, such as carnitinepalmitoyl transferases (e.g., CPT1A, CPT1B, and CPT1C), acyl-CoAoxidases (e.g., ACOX1, ACOX2, and ACOX3), and uncoupling protein (e.g.,UCP2). The data further demonstrate the ability of BCG to attenuateendogenous levels of lipogenic factors, such as acetyl-CoA carboxylases(e.g., ACACA and ACACB), fatty acid synthase, stearoyl-CoA desaturase,malic enzyme, and glucose-6-phosphate dehydrogenase. BCG administrationis additionally capable of promoting a decrease in the level ofadiponectin receptors, such as ADIPOR1 and ADIPOR2, which modulateglucose metabolism and fatty acid oxidation.

FIG. 7A is a graph showing the ability of intradermally-administered BCG(circles) to stabilize and lower cholesterol levels in human subjectsover the course of an 8-year investigation relative to subjects nottreated with BCG (squares). FIG. 7B is a graph showing the ability ofBCG to promote the expression of nuclear receptor subfamily 1 group Hmember 3 (NR1H3) in cultured human peripheral blood lymphocytes (left)and in vivo in human subjects with type 1 diabetes (right). Culturedcells were monitored for NR1H3 expression prior to and 48 hoursfollowing exposure to BCG. Human subjects were assessed for NR1H3expression prior to and 8 weeks following BCG administration. FIG. 7C isa graph showing the ability of BCG to promote the expression of thecholesterol-suppressing genes adenosine triphosphate binding cassettesubfamily A member 1 (ABCA1), adenosine triphosphate binding cassettesubfamily G (ABCG), apolipoprotein E(APOE), Fas cell surface deathreceptor (FAS), and stearoyl-CoA desaturase (SCD1) and reduce theexpression of the glucose-elevating genes fructose-bisphosphatase 1(FBP1), glucose-6-phosphate dehydrogenase (G6PD), and muscle pyruvatekinase (PKM) in human type-1 diabetes patients. Taken together, thesedata demonstrate the capacity of BCG to confer multiple beneficialeffects by suppressing total cholesterol and blood glucoseconcentrations.

FIG. 8A is a graph showing the ability of BCG to lower glycatedhemoglobin levels relative to untreated and to placebo-treated subjectsin long-term human type-1 diabetic patients receiving two doses of BCGseparated by 4 weeks. FIG. 8B is a graph demonstrating the ability ofBCG to promote a decrease in glycated hemoglobin levels in hyperglycemichuman patients and to stabilize glycated hemoglobin near normalphysiologic levels.

FIG. 9A is a schematic depicting the metabolic conversion from oxidativephosphorylation to a state of aerobic glycolysis induced by BCG. FIGS.9B-9H are a series of graphs showing the ability of BCG to induce anincrease in flux through glycolysis (as shown, for instance, by anincrease in lactate levels) in human patients suffering from type-1diabetes. The glucose-suppressing effect of BCG is independent ofpancreas regeneration, as BCG did not promote an increase in the rate ofpurine biosynthesis as assessed by monitoring adenine,N6-carbamoylthreonyadenosine, and methylguanine. FIG. 9I is a tablereporting p-values for differences in metabolite levels for each ofFIGS. 9B-9H. FIGS. 9J and 9K are graphs showing the ability of BCG toenhance hypoxia-inducible factor 1-α (HIF1-α) expression in BCG-treatedperipheral blood lymphocytes in vitro and in BCG-treated monocytes invitro. The conversion from a state of oxidative phosphorylation to astate of aerobic glycolysis is dictated in part by HIF1-α. Takentogether, these data demonstrate the ability of BCG to potentiate thisconversion by up-regulating HIF1-α expression.

FIG. 10A is a schematic illustrating the metabolism of glucose by thepentose phosphate shunt, glycolysis, and the Krebs cycle. FIGS. 10B-10Dare a series of graphs demonstrating the ability of BCG to up-regulatethe glycolytic enzymes hexokinase 2 (HK2), glucose-6-phosphate isomerase(G6PI), triosephosphate isomerase 1 (TPI1), galactokinase 1 (GALK1), andgalactose mutarotase (GALM), as well as the glucose transporter solutecarrier family 2 member 6 (SLC2A6), as well as to modulate theexpression of various proteins involved in the Krebs cycle. Geneexpression data are from mRNA quantitation experiments conducted usingcultured peripheral blood lymphocytes. Taken together, these datademonstrate the ability of BCG to promote processes that result in sugaruptake and glucose consumption.

FIGS. 11A-11D are a series of graphs demonstrating the ability of BCG tomodulate weight and blood sugar levels in hyperglycemic mouse models.Normal BALB/c mice (FIGS. 11A and 11C) were treated with or without BCG.For normal mice, this administration had no consequences on blood sugarlevel or on weight. Weight was measured in these investigations sinceelevated blood sugar levels are associated with weight loss. Chemicallyinduced hyperglycemic BALB/c mice (FIGS. 11B and 11D) were treated witheither BCG or saline. Following BCG administration, hyperglycemic miceexhibited an improved ability to maintain weight (FIG. 11B) and reducedblood glucose relative to mice not treated with BCG (FIG. 11D).Hyperglycemia was induced in BALB/c mice by administration ofstreptozotocin, an agent that non-specifically elevates blood glucose.This is important, as the mice treated in this study were not sufferingfrom a specific disease, but were rather suffering from a non-specificincrease in blood sugar. Taken together, these data demonstrate theability of BCG to reduce blood glucose concentrations in hyperglycemicsubjects in any disease state, regardless of the underlying etiology.FIG. 11E is a graph showing the ability of BCG to reduce glycatedhemoglobin in streptozotocin-treated BALB/c mice. As glycated hemoglobinis an indicator of total blood glucose, these data further demonstratethe ability of BCG to reduce blood glucose concentrations in subjectsexhibiting elevated blood sugar regardless of the underlying biochemicalcause.

DETAILED DESCRIPTION

The invention provides methods of treating diseases, such as thoseassociated with elevated levels of cholesterol, by the administration ofBacillus Calmette-Guerin (BCG). Additionally, a variety of otherdiseases, such as autoimmune diseases, neurological conditions,allergies, allograft rejections, graft-versus-host diseases, asthma,macular degeneration, muscular atrophy, diseases related to miscarriage,atherosclerosis, bone loss, musculoskeletal diseases, and obesity can betreated according to the methods of the invention by administering aneffective amount of BCG to a subject suffering from any of thesediseases.

Methods of the invention also encompass procedures for diagnosing adisease in a subject and for determining whether a subject having aparticular disease is likely to respond to treatment with a therapeuticagent (e.g., BCG) for the disease or would benefit from subsequentdosing of the therapeutic agent (e.g., BCG). The methods involveassessing the presence or level of one or more biomarkers in a subject(e.g., the level of cholesterol or blood glucose or analysis of DNAmethylation patterns in the nuclear DNA isolated from a subject and/ordetermining the levels of one or more mRNA molecules, proteins, and/ormetabolites in a sample isolated from the subject).

Diagnostic Methods of the Invention Assessing Cytosine Methylation State

The invention is based in part on the discovery that BCG modulates themethylation and demethylation of cytosine residues within variousendogenous DNA molecules, particularly DNA molecules within the nucleargenome of a mammalian cell. Cytosine residues that are adjacent to aguanine (e.g., a cytosine-guanine (CG, also referred to as CpG)dinucleotide are susceptible to methylation by DNA methyltransferases atthe C-5 position of the cytosine nucleobase. Co-factors capable ofdonating a methyl group to the cytosine ring include S-adenosylmethionine (SAM), as shown in the biosynthetic scheme below:

BCG is capable of modulating the methylation and demethylation ofcytosine residues (e.g., increasing or decreasing the quantity ofmethylated cytosine residues within a particular DNA sequence),particularly within genes that encode transcription factors, such asFoxP3, or various cell-surface proteins, such as CD45. For instance, theadministration of BCG to a subject may increase or decrease the quantityof methylated cytosine residues in a given DNA sequence, e.g., by about5% or more (e.g., about 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%,35%, 45%, 50%, or more) or by about 1.1-fold or more (e.g., about1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold,1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold,8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold,70-fold, 80-fold, 90-fold, 100-fold, 500-fold, 1,000-fold, 10,000-fold,or more) over the course of one or more hours, days, weeks, months, oryears.

A physician of skill in the art can determine whether a patient (e.g., apatient that has already been diagnosed as having a particular disease,such as elevated cholesterol, LDLs, or triglycerides, reduced HDLlevels, a disease associated with these altered serum lipid levels, oran immunological, neurological, or metabolic disease described herein)is likely to respond to BCG therapy by determining the quantity ofmethylated cytosine residues in a sample isolated from the subject andcomparing this quantity to the amount of methylated cytosine residues inthe DNA sequence of the same genetic locus in a reference sample. Thereference sample may be a sample isolated from a healthy patient,optionally of the same age, sex, and/or weight, or the reference samplemay be a standard quantity of methylated cytosine residues in aparticular DNA sequence that is generally associated with a healthyphysiological state or observed in healthy subjects, such as between 1and 100 methylated cytosine residues (e.g., between 1 and 50 methylatedcytosine residues, between 1 and 25 methylated cytosine residues, orbetween 1 and 10 methylated cytosine residues per molecule of DNA). Adetermination that the quantity of methylated cytosine residues in thesample isolated from the patient is greater than or less than the amountof methylated cytosine residues in the same DNA sequence within areference sample (e.g., by about 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%,30%, 35%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%,or more, or by about 1.1-fold or more, such as about 1.1-fold, 1.2-fold,1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold,2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold,20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold,100-fold, 500-fold, 1,000-fold, 10,000-fold, or more) indicates that thesubject is likely to respond to treatment with a therapeutic agent, suchas BCG, in order to treat the disease. In preferred embodiments, thegene that is analyzed encodes a transcription factor, such as FoxP3, ora cell-surface protein, such as CD45. In these cases, a determinationthat the quantity of methylated cytosine residues in the sample isolatedfrom the subject is greater than the quantity of methylated cytosineresidues in the same DNA sequence within a reference sample (e.g., byabout 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more, or by about1.1-fold or more, such as about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold,1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold,4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold,30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold,500-fold, 1,000-fold, 10,000-fold, or more) indicates that the subjectis likely to respond to administration of a therapeutic agent, such asBCG, in order to treat the disease.

Methods of determining cytosine methylation state in a DNA sequence areknown in the art and often include chemically modifying DNA from thesubject and reference samples in order to convert unmethylated cytosineresidues to uracil by incubating the DNA of interest with bisulfite.Bisulfite sequencing exploits the preferential deamination of cytosinebases to uracil bases in the presence of sodium hydroxide (NaOH) andsodium bisulfite. Methylated cytosine bases (5-methylcytosine), ifpresent, are found almost exclusively at the cytosine position of a CGdinucleotide pair (e.g., 5′-CG-3′). Under acidic conditions, sodiumbisulfite preferentially deaminates cytosine to uracil in a nucleophilicattack while the methyl group on 5-methylcytosine protects the aminogroup from the deamination. As a result, methylated cytosine is notconverted under these conditions. Accordingly, the DNA's originalmethylation state can be analyzed by sequencing the bisulfite convertedDNA and comparing the cytosine position of each cytosine-guanine (CG)dinucleotide pair of an unconverted nucleic acid to bases at thecorresponding positions in the sequence of a bisulfite converted nucleicacid of interest. The cytosine position of a cytosine-guanine (CG)dinucleotide pair of the unconverted nucleic acid is identified ashaving been unmethylated if the corresponding position in the sequenceof a bisulfite converted nucleic acid of interest is now occupied bythymine. The cytosine position of a cytosine-guanine (CG) dinucleotidepair of the unconverted nucleic acid is identified as having beenmethylated if the corresponding position in the sequence of a bisulfiteconverted nucleic acid of interest is occupied by cytosine. Exemplaryprotocols for determining the methylation state of cytosine residues ina DNA molecule of interest are described, e.g., in U.S. Pat. Nos.8,577,615; 7,851,154; WO 2014/149356; and WO 2015/014759; thedisclosures of each of which are incorporated herein by reference.

Analysis of mRNA and Protein Levels

In addition to modulating cytosine methylation state, BCG administrationis capable of modulating the levels of various mRNA molecules andproteins, such as those associated with lipid and glucose metabolism andwith regulating histone acetylation state. For instance, administrationof BCG to a subject is capable of increasing the level of variouscytokines, such as interleukin-6 (IL-6), tumor necrosis factor (TNFα),and interferon-gamma (IFNγ), as well as the mRNA molecules that encodethese proteins. BCG is additionally capable of increasing the levels oflipolytic proteins in a subject, such as acyl co-enzyme A oxidase,carnitine palmitoyltransferase, lipase, and uncoupling protein, as wellas the mRNA molecules that encode these proteins, e.g., by about 5%, 6%,7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 100%, or more, or by about 1.1-fold or more(e.g., about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold,1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold,7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold,60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 500-fold, 1,000-fold,10,000-fold, or more) relative to the quantity of these substances in areference sample, such as a sample isolated from a subject prior toadministration of BCG. Additionally, BCG administration reduces thelevels of adiponectin receptors (e.g., adiponectin receptors 1 and 2)and lipogenic proteins, such as acetyl co-enzyme A carboxylase α, acetylco-enzyme A carboxylase β, fatty acid synthase,glyceraldehydes-6-phosphate dehydrogenase, stearoyl-CoA saturase, malicenzyme, and glucose-6-phosphate dehydrogenase, as well as the mRNAmolecules that encode these proteins. BCG administration is additionallycapable of decreasing the levels of one or more lysineacetyltransferases (KATs), such as KAT2A, KAT2B, KAT5, KAT6A, KAT6B,KAT7, and KAT8, as well as histone acetyltransferases and histonedeacetylases (HDACs, such as HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6,HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, HDAC1P1, HDAC1P2, SIRT1, SIRT2,SIRT3, SIRT4, SIRT5, SIRT6, and SIRT7) and histones (e.g., H2A, H2B, H3,and H4), as well as the mRNA molecules that encode these proteins, e.g.,by about 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more, or by about1.1-fold or more (e.g., about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold,1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold,4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold,30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold,500-fold, 1,000-fold, 10,000-fold, or more) relative to the quantity ofthese substances in a reference sample, such as a sample isolated from asubject prior to administration of BCG.

A physician of skill in the art can determine whether a patient (e.g., apatient that has already been diagnosed as having a particular disease,such as elevated cholesterol, LDLs, or triglycerides, reduced HDLlevels, a disease associated with these altered serum lipid levels, oran immunological, neurological, or metabolic disease described herein)is likely to respond to BCG therapy by determining the quantity of oneor more mRNA molecules or proteins in a sample isolated from the subjectand comparing this quantity to the amount of the same mRNA molecule orprotein in a reference sample. The reference sample may be a sampleisolated from a healthy patient, optionally of the same age, sex, and/orweight, or the reference sample may be a standard concentration of themRNA molecule or protein being analyzed that is generally associatedwith a healthy physiological state or observed in healthy subjects, suchas between 1 pM and 10 mM (e.g., between 1 nM and 100 PM, between 1 nMand 10 PM, or between 1 nM and 1 μM). For instance, the reference samplemay contain between 1 and 100,000 copies, or more, of an mRNA transcript(e.g., an mRNA transcript described herein). A reference sample maycontain one or more cells, such as a cell of the hematopoietic lineage(e.g., a CD4+, CD25+T-reg cell) that each contain between 1 and 100,000copies, or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40,50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000,5,000, 10,000, 50,000, or 100,000 copies, or more) of an mRNA transcriptof interest, such as an mRNA transcript that encodes the CD45 or FoxP3proteins. A determination that the quantity of lipolytic proteins orcytokines, such as those described herein, or the mRNA molecules thatencode any one of these proteins, in the sample isolated from thepatient is less than (e.g., by about 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%,25%, 30%, 35%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,100%, or more, or by about 1.1-fold or more, such as by about 1.1-fold,1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold,1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold,9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold,80-fold, 90-fold, 100-fold, 500-fold, 1,000-fold, 10,000-fold, or more)the amount of the same lipolytic protein or cytokine within a referencesample indicates that the subject is likely to respond to treatment witha therapeutic agent, such as BCG, in order to treat the disease.Conversely, a determination that the quantity of lipogenic proteins,adiponectin receptors, KATs, histone acetyltransferases, HDACs, orhistones, such as those described herein, or the mRNA molecules thatencode any one of these proteins, in the sample isolated from thepatient is greater than (e.g., by about 5%, 6%, 7%, 8%, 9%, 10%, 15%,20%, 25%, 30%, 35%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 100%, or more, or by about 1.1-fold or more, such as by about1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold,1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold,8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold,70-fold, 80-fold, 90-fold, 100-fold, 500-fold, 1,000-fold, 10,000-fold,or more) the amount of the same protein or mRNA molecule within areference sample indicates that the subject is likely to respond totreatment with a therapeutic agent, such as BCG, in order to treat thedisease.

Methods for determining the concentration of proteins are known in theart and include, without limitation, ELISA-based assays, immunoblotassays, such as Western blot experiments, as well as HPLC, massspectrometry, and UV-Vis spectroscopy, among others.

Standard methods for the detection and quantitation of mRNA moleculesare additionally known in the art. Exemplary techniques for thisanalysis include Northern blot experiments and quantitativereverse-transcription polymerase chain reaction (PCR) techniques. Usingthis technique, it is possible to determine the quantity of a targetmRNA molecule in a sample (e.g., in lysate obtained from a population ofcells isolated from a patient, such as from the blood of a patient) byfirst reverse-transcribing the target mRNA to produce cDNA moleculesencoding the protein that is obtained by translation of the target mRNAsequence. This technique involves lysing a population of cells isolatedfrom a patient (e.g., T-lymphocytes, such as T-reg cells) that containone or more copies of an mRNA molecule of interest. Optionally, thelysis can be performed in the presence of a chaotropic agent, such asbetween about 0.05 M and 1 M of a chaotropic agent. These are substancescapable of disrupting the three dimensional structure of macromoleculessuch as proteins, DNA, or RNA and denatures them. Chaotropic agentsinterfere with stabilizing intramolecular interactions mediated bynon-covalent forces such as hydrogen bonds, Van der Waals forces, andhydrophobic effects. Chaotropic reagents include but are not limited tourea, various lithium salts such lithium perchlorate, and guanidiniumsalts, such as guanidinium chloride. The cDNA is subsequently amplifiedusing standard thermocycling techniques, e.g., as described in U.S. Pat.No. 8,623,602, the disclosure of which is incorporated herein byreference.

In certain cases, it may be advantageous to remove genomic DNA from acell lysate to avoid the possibility of amplifying endogenous DNA incombination with cDNA during the thermocycling procedure. To this end,an effective technique for the removal of genomic DNA is enzymaticdigestion. This can be achieved by treating the lysate sample with aDNAse, such as DNAse I or Shrimp DNAse, e.g., as disclosed in U.S. Pat.No. 6,541,204, the disclosure of which is incorporated herein byreference. In these cases, the DNAse is desirably inactivated followingthe degradation of genomic cDNA so as to prevent enzymatic cleavage ofthe cDNA synthesized from the target mRNA molecule. DNAse inactivationcan be achieved, e.g., by incubating the reaction mixture containing theDNAse and the cell lysate at an elevated temperature, such as at between80° C. and 90° C. for about 10 minutes.

To amplify the first cDNA strand from a target m RNA molecule, a primercomplimentary to a sequence located at the 3′ end of the target mRNAmolecule can be designed, e.g., based on sequence elements present inmammalian mRNAs, such as poly-adenyl (pA) tails. Oligomericdeoxyribothymidine (dT) primers, which site-specifically bind to the pAtail of an mRNA, can be used as a primer to initiate the synthesis ofthe initial cDNA strand. To synthesize the subsequent cDNA strand, aprimer complimentary to the 5′ end of the initial cDNA strand isdesirably included in the reaction mixture. This primer may be, e.g., aprimer of between 10 and 30 nucleotides in length that has thecorresponding DNA sequence of the target mRNA molecule in a 5′-to-3′direction. Examples of RNA dependent DNA polymerases that can be usedfor these cDNA synthesis steps include AMV Reverse Transcriptase (RocheApplied Science Cat. No. 11 495 062), MMuLV Reverse Transcriptase (RocheApplied Science Cat No. 011 062 603), and the recombinant TranscriptorReverse Transcriptase (Roche Applied Science Cat. No. 03 531 317).Subsequently, all reagents are added that are required to amplify thegenerated single stranded cDNA by means of PCR, such as a thermostableDNA dependent DNA polymerases as well as target-specific forward andreverse amplification primers. The amplified DNA sequence is thenquantitated using one of a variety of techniques, such as fluorescenceresonant energy transfer (FRET)-based techniques, described in detail,e.g., in U.S. Pat. Nos. 5,210,015; 5,538,848; 5,487,972; 5,804,375,5,118,801; WO 1997/046707; WO 1997/046712; and WO 1997/046714, thedisclosures of each of which are incorporated herein by reference.

Determination of Amino Acid and Metabolite Levels

In addition to modulating mRNA and protein concentrations, BCGadministration is capable of regulating the levels of various acetylatedamino acids and methylated metabolites. For instance, administration ofBCG to a subject is capable of increasing the level of variousN-acetylated amino acids, such as N-acetylalanine, N-acetylasparticacid, N-acetylserine, N-acetylthreonine, N-acetylhistidine,N-acetyl-3-methylhistidine, N-acetylvaline, and N-α-acetyllysine, andN-acetylmethionine (e.g., by promoting acetylation of alanine, asparticacid, serine, threonine, histidine, 3-methylhistidine, valine, lysine,and methionine) e.g., by about 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%,30%, 35%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%,or more, or by about 1.1-fold or more, such as by about 1.1-fold,1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold,1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold,9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold,80-fold, 90-fold, 100-fold, 500-fold, 1,000-fold, 10,000-fold, or more,relative to the quantity of these substances in a reference sample, suchas a sample isolated from a subject prior to administration of BCG. BCGis additionally capable of increasing the levels of various methylatedmetabolites in a subject, such as N-α-acetyl-3-methylhistidine,3-methylglutaconic acid, 3-methylglutarylcarnitine, andN-ε-trimethyllysine (e.g., by promoting the methylation ofN-α-acetylhistidine, glutaconic acid, glutarylcarnitine, lysine, andcysteine), e.g., by about 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%,35%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, ormore, or by about 1.1-fold or more, such as by about 1.1-fold, 1.2-fold,1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold,2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold,20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold,100-fold, 500-fold, 1,000-fold, 10,000-fold, or more, relative to thequantity of these substances in a reference sample, such as a sampleisolated from a subject prior to administration of BCG.

Additionally, BCG administration reduces the levels of certainmethylated metabolites, such as 4-methyl-2-oxopentanoic acid and3-methyl-2-oxobutyric acid (e.g., by promoting demethylation of thesemetabolites) e.g., by about 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%,35%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, ormore, or by about 1.1-fold or more, such as by about 1.1-fold, 1.2-fold,1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold,2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold,20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold,100-fold, 500-fold, 1,000-fold, 10,000-fold, or more, relative to thequantity of these substances in a reference sample, such as a sampleisolated from a subject prior to administration of BCG.

A physician of skill in the art can determine whether a patient (e.g., apatient that has already been diagnosed as having a particular disease,such as elevated cholesterol, LDLs, or triglycerides, reduced HDLlevels, a disease associated with these altered serum lipid levels, oran immunological, neurological, or metabolic disease described herein)is likely to respond to BCG therapy by determining the quantity of oneor more acetylated amino acids or methylated metabolites in a sampleisolated from the subject and comparing this quantity to the amount ofthe same substance in a reference sample. The reference sample may be asample isolated from a healthy patient, optionally of the same age, sex,and/or weight, or the reference sample may be a standard concentrationof the amino acid or metabolite being analyzed that is generallyassociated with a healthy physiological state or observed in healthysubjects, such as between 1 pM and 10 mM (e.g., between 1 nM and 100 PM,between 1 nM and 10 PM, or between 1 nM and 1 μM). A determination thatthe quantity of N-acetylalanine, N-acetylaspartic acid, N-acetylserine,N-acetylthreonine, N-acetylhistidine, N-acetyl-3-methylhistidine,N-acetylvaline, and N-α-acetyllysine, N-acetylmethionine,N-α-acetyl-3-methylhistidine, 3-methylglutaconic acid,3-methylglutarylcarnitine, and/or N-ε-trimethyllysine in the sampleisolated from the patient is less than the amount of the same substancewithin a reference sample (e.g., by about 5%, 6%, 7%, 8%, 9%, 10%, 15%,20%, 25%, 30%, 35%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 100%, or more, or by about 1.1-fold or more, such as by about1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold,1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold,8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold,70-fold, 80-fold, 90-fold, 100-fold, 500-fold, 1,000-fold, 10,000-fold,or more) indicates that the subject is likely to respond to treatmentwith a therapeutic agent, such as BCG, in order to treat the disease.Conversely, a determination that the quantity of 4-methyl-2-oxopentanoicacid and/or 3-methyl-2-oxobutyric acid in the sample isolated from thepatient is greater than the amount of the same substance within areference sample (e.g., by about 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%,30%, 35%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%,or more, or by about 1.1-fold or more, such as by about 1.1-fold,1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold,1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold,9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold,80-fold, 90-fold, 100-fold, 500-fold, 1,000-fold, 10,000-fold, or more)indicates that the subject is likely to respond to treatment with atherapeutic agent, such as BCG, in order to treat the disease.

Methods for determining the concentration of acetylated amino acids andmethylated metabolites are known in the art and include, withoutlimitation, nuclear magnetic resonance (NMR) spectroscopy, HPLC, massspectrometry, and UV-Vis spectroscopy, among others.

Biomarkers for Diagnosing a Patient as Having a Disease

In addition to determining whether a patient is likely to respond totherapy (e.g., by administration of BCG), the methods of the inventioncan also be used to render a diagnosis of a particular disease (e.g., animmunological or neurological condition, or a disease associated with anelevated level of serum cholesterol, as described herein). For instance,a physician of skill in the art may analyze the level of cytosinemethylation in a nuclear gene of interest in a sample isolated from asubject (e.g., a blood sample, such as a blood sample containing one ormore T-reg cells from which the DNA is isolated and analyzed) in orderto determine if the subject has a particular disorder. The quantity ofmethylated cytosine residues in a nuclear gene isolated from one or morecells of the sample can then be compared to the quantity of methylatedcytosine residues in the same gene isolated from a reference sample. Thereference sample may be a sample isolated from a healthy patient,optionally of the same age, sex, and/or weight as the subject beingassessed. Alternatively, the reference sample may be a standard quantityof methylated cytosine residues in a particular DNA sequence that isgenerally associated with a healthy physiological state or observed inhealthy subjects, such as between 1 and 100 methylated cytosine residues(e.g., between 1 and 50 methylated cytosine residues, between 1 and 25methylated cytosine residues, or between 1 and 10 methylated cytosineresidues per molecule of DNA). A determination that the quantity ofmethylated cytosine residues in the sample isolated from the patient isgreater than or less than the amount of methylated cytosine residues inthe same DNA sequence within a reference sample (e.g., by about 5%, 6%,7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 100%, or more, or by about 1.1-fold or more,such as about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold,1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold,6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold,50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 500-fold,1,000-fold, 10,000-fold, or more) indicates that the subject may have aparticular disease, such as an immunological or neurological disease, ora disorder associated with an elevated serum cholesterol concentration,as described herein. This determination may alternatively indicate thatthe patient would benefit from administration of BCG to prevent theonset of such a disease or condition. The gene that is analyzed mayencode a transcription factor, such as FoxP3, or a cell-surface protein,such as CD45. In these cases, a determination that the quantity ofmethylated cytosine residues in the sample isolated from the subject isgreater than the quantity of methylated cytosine residues in the sameDNA sequence within a reference sample (e.g., by about 5%, 6%, 7%, 8%,9%, 10%, 15%, 20%, 25%, 30%, 35%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 100%, or more, or by about 1.1-fold or more, such asabout 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold,1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold,7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold,60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 500-fold, 1,000-fold,10,000-fold, or more) indicates that the subject may have aimmunological, neurological, or cholesterol-related disorder. Thisdetermination may alternatively indicate that the patient would benefitfrom administration of BCG to prevent the onset of such a disease orcondition.

In addition to rendering a diagnosis of a particular disease on thebasis of cytosine methylation state, the methods of the inventionadditionally provide procedures for diagnosing a patient as having animmunological, neurological, or cholesterol-related disorder based onthe levels of various mRNA molecules and proteins, such as thoseassociated with lipid and glucose metabolism and with regulating histoneacetylation state. For instance, a physician of skill in the art can usethe methods of the invention to measure the level of one or morecytokines, such as IL-6, TNFα, and IFNγ, as well as the mRNA moleculesthat encode these proteins, in order to diagnose a patient as having aparticular disorder. A physician may additionally or alternativelymonitor the level of one or more lipolytic proteins in a subject, suchas acyl co-enzyme A oxidase, carnitine palmitoyltransferase, lipase, anduncoupling protein, as well as the mRNA molecules that encode theseproteins. A determination that the quantity of one or more of theselipolytic proteins or cytokines, or one or more of the mRNA moleculesthat encode these proteins, in the sample isolated from the patient isless than (e.g., by about 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%,35%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, ormore, or by about 1.1-fold or more, such as by about 1.1-fold, 1.2-fold,1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold,2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold,20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold,100-fold, 500-fold, 1,000-fold, 10,000-fold, or more) the amount of thesame lipolytic protein or cytokine within a reference sample (such as asample isolated from a healthy subject as described above) indicatesthat the subject being assessed may have an immunological, neurological,or cholesterol-related disorder as described herein. Conversely, adetermination that the quantity of lipogenic proteins, adiponectinreceptors, KATs, histone acetyltransferases, HDACs, or histones, such asthose described herein, or the mRNA molecules that encode any one ofthese proteins, in the sample isolated from the patient is greater than(e.g., by about 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more, or byabout 1.1-fold or more, such as by about 1.1-fold, 1.2-fold, 1.3-fold,1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold,3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold,20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold,100-fold, 500-fold, 1,000-fold, 10,000-fold, or more) the amount of thesame protein or mRNA molecule within a reference sample indicates thatthe subject may have an immunological, neurological, orcholesterol-related disorder as described herein. These determinations,individually or collectively, may alternatively indicate that thepatient would benefit from administration of BCG to prevent the onset ofsuch a disease or condition.

A physician of skill in the art can also determine whether a patient hasa particular disease (e.g., a disease associated with elevatedcholesterol, LDLs, or triglycerides, reduced HDL levels, a diseaseassociated with these altered serum lipid levels, or an immunological,neurological, or metabolic disease described herein) by determining thequantity of one or more acetylated amino acids or methylated metabolitesin a sample isolated from the subject and comparing this quantity to theamount of the same substance in a reference sample. As described for themethods of diagnosis on the basis of DNA methylation patterns andmRNA/protein levels, the reference sample may be a sample isolated froma healthy patient, optionally of the same age, sex, and/or weight, orthe reference sample may be a standard concentration of the amino acidor metabolite being analyzed that is generally associated with a healthyphysiological state or observed in healthy subjects, such as between 1pM and 10 mM (e.g., between 1 nM and 100 μM, between 1 nM and 10 μM, orbetween 1 nM and 1 μM). A determination that the quantity ofN-acetylalanine, N-acetylaspartic acid, N-acetylserine,N-acetylthreonine, N-acetylhistidine, N-acetyl-3-methylhistidine,N-acetylvaline, and N-α-acetyllysine, N-acetylmethionine,N-α-acetyl-3-methylhistidine, 3-methylglutaconic acid,3-methylglutarylcarnitine, and/or N-ε-trimethyllysine in the sampleisolated from the patient is less than the amount of the same substancewithin a reference sample (e.g., by about 5%, 6%, 7%, 8%, 9%, 10%, 15%,20%, 25%, 30%, 35%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 100%, or more, or by about 1.1-fold or more, such as by about1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold,1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold,8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold,70-fold, 80-fold, 90-fold, 100-fold, 500-fold, 1,000-fold, 10,000-fold,or more) indicates that the subject may have an immunological,neurological, or cholesterol-related disease, e.g., as described herein.Conversely, a determination that the quantity of 4-methyl-2-oxopentanoicacid and/or 3-methyl-2-oxobutyric acid in the sample isolated from thepatient is greater than the amount of the same substance within areference sample (e.g., by about 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%,30%, 35%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%,or more, or by about 1.1-fold or more, such as by about 1.1-fold,1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold,1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold,9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold,80-fold, 90-fold, 100-fold, 500-fold, 1,000-fold, 10,000-fold, or more)indicates that the subject may have an immunological, neurological, orcholesterol-related disease, e.g., as described herein. Thesedeterminations, individually or collectively, may alternatively indicatethat the patient would benefit from administration of BCG to prevent theonset of such a disease or condition.

In addition to the above, the methods of the invention additionallyprovide procedures for diagnosing a patient as having hyperglycemia or adisease associated with hyperglycemia, such as type-2 diabetes, as wellas methods of predicting whether such patients would benefit from BCGtreatment. I have discovered that BCG is capable of suppressingcholesterol levels, for instance, by increasing the expression of NR1H3,thereby promoting the expression of cholesterol-suppressing genes suchas ABCA1, ABCG, APOE, FAS, and SCD1 and down-regulating the expressionof glucose-elevating genes such as FBP1, G6PD, and PKM. I have alsodiscovered that BCG reduces the expression of enzymes that promote fluxthrough the Krebs cycle, such as ACLY, ACO2, CS, DLD, OGDH, SDHB, andSUCLG1, while enhancing the expression of glycolytic enzymes such asHK2, G6PI, TPI2, GALK1, and GALM, as well as glucose transporters, suchas SLC2A6. BCG additionally increases the expression of HIF1-α, inducingglucose depletion by promoting a transition from oxidativephosphorylation to aerobic glycolysis, which consumes glucose much morerapidly so as to produce adenosine triphosphate. A physician of skill inthe art can use the methods of the invention to measure the level of oneor more the preceding substances, or an mRNA molecule encoding suchsubstances, so as to determine whether a patient has a hyperglycemiccondition or may benefit from BCG therapy. For instance, a determinationthat the quantity of one or more of the preceding enzymes that promotesflux through the Krebs cycle, α-ketobutyrate, 2-hydroxybutyrate, FBP1,G6PD, or PKM is increased (e.g., by about 5%, 6%, 7%, 8%, 9%, 10%, 15%,20%, 25%, 30%, 35%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 100%, or more, or by about 1.1-fold or more, such as by about1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold,1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold,8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold,70-fold, 80-fold, 90-fold, 100-fold, 500-fold, 1,000-fold, 10,000-fold,or more) relative to the amount of the same substance within a referencesample (such as a sample isolated from a healthy subject as describedabove) indicates that the subject being assessed may have hyperglycemiaor a disease associated with hyperglycemia, and/or may benefit from BCGtherapy. Conversely, a determination that the quantity a glycolyticenzyme, glucose transporter, HIF1-α, NR1H3, lactate, or1,5-anhydroglucitol is reduced (e.g., by about 5%, 6%, 7%, 8%, 9%, 10%,15%, 20%, 25%, 30%, 35%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 100%, or more, or by about 1.1-fold or more, such as by about1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold,1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold,8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold,70-fold, 80-fold, 90-fold, 100-fold, 500-fold, 1,000-fold, 10,000-fold,or more) relative to the amount of the substance within a referencesample indicates that the subject may have hyperglycemia or a diseaseassociated with hyperglycemia, and/or may benefit from BCG therapy.These determinations, individually or collectively, may indicate thatthe patient would benefit from subsequent administration of BCG in oneor more additional doses.

Methods of Treatment and Prevention Administration of BCG to ModulateSerum Lipid Levels

The invention provides methods of treating a subject that has one ormore diseases by administering an effective amount of BCG to thesubject. According to the methods of the invention, BCG can beadministered to a subject in order to reduce the level of cholesterol,low-density lipoproteins (LDLs), and/or triglycerides. The subject maybe one that has already been diagnosed as having elevated levels ofcholesterol, LDLs, and/or triglycerides. The subject may also be onethat is prone to development of hypercholesteremia in the future; assuch, BCG can also be used as a prophylactic therapy according to themethods of the invention. Cholesterol levels in a healthy human subjectare typically about 129 mg/dL, and therefore a serum cholesterolconcentration above this threshold may be considered an elevated levelof cholesterol. For instance, a subject that has a serum cholesterollevel of about 135 mg/dL, 140 mg/dL, 145 mg/dL, 150 mg/dL, 155 mg/dL,160 mg/dL, 165 mg/dL, 170 mg/dL, 175 mg/dL, 180 mg/dL, 185 mg/dL, 190mg/dL, 195 mg/dL, 200 mg/dL, or greater, can be considered to haveelevated an level of cholesterol. A subject may be classified as havingan elevated level of LDLs if the subject has an LDL level of about 80mg/dL or greater (e.g., about 80 mg/dL, 85 mg/dL, 90 mg/dL, 95 mg/dL,100 mg/dL, 105 mg/dL, 110 mg/dL, 115 mg/dL, 120 mg/dL, 125 mg/dL, 130mg/dL, 135 mg/dL, 140 mg/dL, 145 mg/dL, 150 mg/dL, or greater). Asubject may be classified as having an elevated level of triglyceridesif the subject has a serum triglyceride level of about 100 mg/dL or more(e.g., about 100 mg/dL, 105 mg/dL, 110 mg/dL, 115 mg/dL, 120 mg/dL, 125mg/dL, 130 mg/dL, 145 mg/dL, 150 mg/dL, 155 mg/dL, 160 mg/dL, 165 mg/dL,170 mg/dL, 175 mg/dL, 180 mg/dL, 195 mg/dL, 200 mg/dL, 205 mg/dL, 210mg/dL, 215 mg/dL, 220 mg/dL, 225 mg/dL, 230 mg/dL, 235 mg/dL, 240 mg/dL,245 mg/dL, 250 mg/dL, 255 mg/dL, 260 mg/dL, 265 mg/dL, 270 mg/dL, 275mg/dL, 280 mg/dL, 285 mg/dL, 290 mg/dL, 300 mg/dL, 305 mg/dL, 310 mg/dL,315 mg/dL, 320 mg/dL, 325 mg/dL, 330 mg/dL, 335 mg/dL, 340 mg/dL, 345mg/dL, 350 mg/dL, 355 mg/dL, 360 mg/dL, 365 mg/dL, 370 mg/dL, 375 mg/dL,380 mg/dL, 385 mg/dL, 390 mg/dL, 400 mg/dL, 405 mg/dL, 410 mg/dL, 415mg/dL, 420 mg/dL, 425 mg/dL, 430 mg/dL, 435 mg/dL, 440 mg/dL, 445 mg/dL,450 mg/dL, 455 mg/dL, 460 mg/dL, 465 mg/dL, 470 mg/dL, 480 mg/dL, 485mg/dL, 490 mg/dL, 495 mg/dL, 500 mg/dL, or greater).

In addition, a subject may be classified as having an elevated level ofcholesterol, LDLs, or triglycerides if the subject currently has a serumlevel of one or more of these substances that is higher than that whichhas previously been observed in a sample isolated from the subject. Forinstance, even though a subject may have a serum cholesterol level ofless than 129 mg/dL (e.g., about 100 mg/dL, 105 mg/dL, 110 mg/dL, 115mg/dL, 120 mg/dL, or 125 mg/dL), the subject can be considered to havean elevated level of cholesterol if the subject has a serum cholesterollevel that is higher than a serum cholesterol level that was previouslyobserved in a sample isolated from the subject (e.g., between about 1day and about 5-20 years ago). The elevated cholesterol level may beincreased, for instance, by about 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 100%, or more relative to the level of serum cholesterol previouslymeasured in a sample from the subject. Likewise, a subject can beconsidered to have an elevated level of serum LDLs if the subject has ahigher serum LDL level that that which has been previously observed in asample from the subject, e.g., even if the subject currently has a serumLDL level that is less than about 80 mg/dL (e.g., about 75 mg/dL, 70mg/dL, 65 mg/dL, 60 mg/dL, 55 mg/dL, 50 mg/dL, 45 mg/dL, 40 mg/dL, orlower). The elevated LDL level may be increased, for instance, by about5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more relative to thelevel of serum LDLs previously measured in a sample from the subject.Additionally, a subject can be considered to have elevated levels ofserum triglycerides if the subject has a higher serum triglyceride levelthat that which has been previously observed in a sample from thesubject, e.g., even if the subject currently has a serum triglyceridelevel that is less than about 100 mg/dL (e.g., about 95 mg/dL, 90 mg/dL,85 mg/dL, 80 mg/dL, 75 mg/dL, 70 mg/dL, 65 mg/dL, 60 mg/dL, 55 mg/dL, 50mg/dL, 45 mg/dL, 40 mg/dL, or lower). The elevated triglyceride levelmay be increased, for instance, by about 5%, 6%, 7%, 8%, 9%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 100%, or more relative to the level of serum triglyceridespreviously measured in a sample from the subject or relative to ahealthy level of serum triglycerides (e.g., less than about 150 mg/dL).

A subject can be considered to be in need of an increase in the level ofserum HDLs if the subject has a serum HDL level of about 40 mg/dL orlower (e.g., about 40 mg/dL, 35 mg/dL, 30 mg/dL, 25 mg/dL, 20 mg/dL, 15mg/dL, or lower). Additionally, a subject can be considered to be inneed of an increase in the level of serum HDLs if the subject has aserum HDL level that is lower than that which has previously beenmeasured in a sample from the subject. For instance, a subject can beconsidered to be in need of an increase in the level of serum HDLs evenif the subject has a serum HDL level that is about 40 mg/dL or greater.It is known in the art that higher HDL levels more effectively protect asubject against developing elevated serum cholesterol and diseasesassociated therewith, such as a disease described herein. A subject inneed of an increase in serum HDLs may have a current serum HDL levelthat is reduced, e.g., by about 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,100%, or more relative to the level of serum HDLs previously measured ina sample from the subject.

According to the methods of the invention, BCG can be administered to asubject in order to decrease the level of cholesterol, LDLs, and/ortriglycerides in the subject. Administration of BCG may reduce the levelof serum cholesterol, LDLs, and/or triglycerides by about 5% or more.For instance, a subject that has been diagnosed as having an elevatedlevel of cholesterol can be administered BCG in order to lower the levelof serum cholesterol in the subject, e.g., by about 5%, 6%, 7%, 8%, 9%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%. 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 100%, or more relative to the subject's level ofserum cholesterol at the time the BCG is administered. BCG mayeffectively reduce the subject's serum cholesterol level to a healthyvalue of about 129 mg/dL or lower, e.g., administration of BCG mayreduce a subject's serum cholesterol level to about 200 mg/dL, 195mg/dL, 190 mg/dL, 185 mg/dL, 180 mg/dL, 185 mg/dL, 180 mg/dL, 175 mg/dL,170 mg/dL, 165 mg/dL, 160 mg/dL, 155 mg/dL, 150 mg/dL, 145 mg/dL, 140mg/dL, 135 mg/dL, 130 mg/dL, 125 mg/dL, 120 mg/dL, 115 mg/dL, 110 mg/dL,105 mg/dL, 100 mg/dL, or lower. Additionally or alternatively, a subjectthat has been diagnosed as having an elevated level of serum LDLs can beadministered BCG in order to lower the level of serum LDLs in thesubject, e.g., by about 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%,or more relative to the subject's level of serum LDLs at the time theBCG is administered. BCG may effectively reduce the subject's serum LDLlevel to a healthy value of about 80 mg/dL or lower, e.g.,administration of BCG may reduce a subject's serum cholesterol level toabout 150 mg/dL, 145 mg/dL, 140 mg/dL, 135 mg/dL, 130 mg/dL, 125 mg/dL,120 mg/dL, 115 mg/dL, 110 mg/dL, 105 mg/dL, 100 mg/dL, 95 mg/dL, 90mg/dL, 85 mg/dL, 80 mg/dL, 75 mg/dL, 70 mg/dL, 65 mg/dL, 60 mg/dL, 55mg/dL, 50 mg/dL, 45 mg/dL, 40 mg/dL, or lower. According to the methodsof the invention, a subject that has been diagnosed as having anelevated level of serum triglycerides can be administered BCG in orderto lower the level of serum triglycerides in the subject, e.g., by about5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%.60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more relative to thesubject's level of serum triglycerides at the time the BCG isadministered. Administration of BCG may effectively reduce the subject'sserum triglyceride level to a healthy value of about 100 mg/dL or lower,e.g., administration of BCG may reduce the subject's serum triglyceridelevel to about 500 mg/dL, 495 mg/dL, 490 mg/dL, 485 mg/dL, 480 mg/dL,475 mg/dL, 470 mg/dL, 465 mg/dL, 460 mg/dL, 455 mg/dL, 450 mg/dL, 445mg/dL, 440 mg/dL, 435 mg/dL, 430 mg/dL, 425 mg/dL, 420 mg/dL, 415 mg/dL,410 mg/dL, 405 mg/dL, 400 mg/dL, 395 mg/dL, 390 mg/dL, 385 mg/dL, 380mg/dL, 375 mg/dL, 370 mg/dL, 365 mg/dL, 360 mg/dL, 355 mg/dL, 350 mg/dL,355 mg/dL, 350 mg/dL, 345 mg/dL, 340 mg/dL, 335 mg/dL, 330 mg/dL, 325mg/dL, 320 mg/dL, 315 mg/dL, 310 mg/dL, 305 mg/dL, 300 mg/dL, 295 mg/dL,290 mg/dL, 285 mg/dL, 280 mg/dL, 275 mg/dL, 270 mg/dL, 265 mg/dL, 260mg/dL, 255 mg/dL, 250 mg/dL, 245 mg/dL, 240 mg/dL, 235 mg/dL, 230 mg/dL,225 mg/dL, 220 mg/dL, 215 mg/dL, 210 mg/dL, 205 mg/dL, 200 mg/dL, 195mg/dL, 190 mg/dL, 185 mg/dL, 180 mg/dL, 175 mg/dL, 170 mg/dL, 165 mg/dL,160 mg/dL, 155 mg/dL, 150 mg/dL, 145 mg/dL, 140 mg/dL, 135 mg/dL, 130mg/dL, 125 mg/dL, 120 mg/dL, 115 mg/dL, 110 mg/dL, 105 mg/dL, 100 mg/dL,95 mg/dL, 90 mg/dL, 85 mg/dL, 80 mg/dL, 75 mg/dL, 70 mg/dL, 65 mg/dL, 60mg/dL, 55 mg/dL, 50 mg/dL, 45 mg/dL, 40 mg/dL, or lower.

Additionally or alternatively, BCG can be administered to a subject thatis prone to develop elevated levels of serum cholesterol, LDLs, and/ortriglycerides, even though the patient may not currently exhibitelevated serum concentrations of these lipids. For instance, BCG can beadministered to a subject that currently has a serum cholesterol levelthat is less than 129 mg/dL (e.g., about 100 mg/dL, 105 mg/dL, 110mg/dL, 115 mg/dL, 120 mg/dL, or 125 mg/dL) and that is not considered topresently have elevated levels of serum cholesterol (e.g., the subject'scurrent serum cholesterol level is within the range (e.g., within10%-25%) of a cholesterol level previously observed for the subject).The administration of BCG may prevent the subject from developing, ormay reduce the likelihood that the subject will develop, elevated serumcholesterol levels, such that upon future examination of the subject,e.g., between about 1 day and 20 years, or more, following theadministration (such as about 1 day, 1 week, 1 month, 6 months, 1 year,2 years, 3 years, 4 years, 5 years, 10 years, 15 years, or 20 years, ormore, following the administration of BCG), the subject may presentserum cholesterol levels that are not considered elevated (e.g., basedon the description of an elevated cholesterol level described above. Inthe same manner, BCG can be used as a prophylactic therapy forpreventing the development of elevated serum LDL and/or triglyceridelevels, such that following the administration of BCG, the subject mayexhibit serum LDL and/or triglyceride levels that are not consideredelevated (e.g., based on the descriptions of elevated serum LDLs andtriglycerides described above). Prophylactic BCG therapy may beindicated in a subject that has a family history of, or a geneticpredisposition to develop, elevated levels of serum cholesterol, LDLs,and/or triglycerides.

The methods of the invention can also be used to elevate the level ofserum HDLs in a subject by administration of BCG. According to thesemethods, the BCG may induce an increase in the level of serum HDLs,e.g., by about 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or morerelative to the subject's level of serum HDLs at the time the BCG isadministered. Administration of BCG may effectively increase thesubject's serum HDL level to a healthy value of about 40 mg/dL, e.g.,administration of BCG may increase the subject's serum HDL level toabout 40 mg/dL, 45 mg/dL, 50 mg/dL, 55 mg/dL, 60 mg/dL, 65 mg/dL, 70mg/dL, 75 mg/dL, 80 mg/dL, 85 mg/dL, 90 mg/dL, 95 mg/dL, 100 mg/dL, orgreater.

Additionally or alternatively, BCG can be administered to a subject thatis prone to develop attenuated levels of serum HDLs, even though thepatient may not currently exhibit decreased serum concentrations ofthese lipids. For instance, BCG can be administered to a subject thatcurrently has a serum HDL level that is between about 40 mg/dL and about80 mg/dL (e.g., about 40 mg/dL, 45 mg/dL, 50 mg/dL, 55 mg/dL, 60 mg/dL,65 mg/dL, 70 mg/dL, 75 mg/dL, and 80 mg/dL) and that is not consideredto presently be in need of an increase in the level of serum HDLs (e.g.,the subject's current serum HDL level is within the range (e.g., within10%-25%) of a serum HDL level previously observed for the subject). Theadministration of BCG may prevent the subject from developing decreasedserum HDL level, such that upon future examination of the subject, e.g.,between about 1 day and 20 years, or more, following the administration(such as about 1 day, 1 week, 1 month, 6 months, 1 year, 2 years, 3years, 4 years, 5 years, 10 years, 15 years, or 20 years, or more,following the administration of BCG), the subject may exhibit serum HDLlevels that are not considered attenuated (e.g., the subject may not bein need of an increased HDL level as described above). Prophylactic BCGtherapy may be indicated in a subject that has a family history of, or agenetic predisposition to develop, attenuated (e.g., lower) levels ofserum HDLs.

In addition to methods of modulating serum lipid levels, the inventionprovides methods of treating a variety of diseases associated withelevated serum cholesterol, LDL, and/or triglyceride concentrations. Forinstance, BCG can be administered to a subject in order to treathypercholesterolemia, hyperlipidemia, coronary heart disease, peripheralarterial disease (PAD), peripheral vascular disease, hypertension,stroke, diabetes, metabolic syndrome, obesity, and/or insulin resistancein the subject. BCG administration can also be performed according tothe methods of the invention in order to alleviate one or more symptomsassociated with such diseases, including elevated levels of lactatedehydrogenase (LDH), LDL, and triglycerides in the subject. Forinstance, BCG administration may be capable of reducing LDH levels in asubject, e.g., by about 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%,or more relative to the subject's level of serum HDLs at the time theBCG is administered. Additional symptoms that can be alleviated byadministration of BCG include angina, arrhythmia, and heart failure. BCGcan also be administered as a prophylactic to prevent these diseases andsymptoms associated therewith or to reduce the likelihood that thesediseases and/or one or more of their symptoms may develop. The sectionsbelow provide a description of exemplary diseases that can be treatedand prevented by administration of BCG.

Effect of BCG on NR1H3 Expression

The present invention is based in part on the discovery that BCG iscapable of promoting the expression of nuclear receptor subfamily 1group H member 3 (NR1H3). NR1H3 is a nuclear receptor protein capable ofup-regulating the expression of cholesterol-suppressing genes, such asadenosine triphosphate binding cassette subfamily A member 1 (ABCA1),adenosine triphosphate binding cassette subfamily G (ABCG),apolipoprotein E(APOE), Fas cell surface death receptor (FAS), andstearoyl-CoA desaturase (SCD1). NR1H3 additionally reduces theexpression of glucose-elevating genes, such as fructose-bisphosphatase 1(FBP1), glucose-6-phosphate dehydrogenase (G6PD), and muscle pyruvatekinase (PKM). As demonstrated in FIGS. 7A-7C, BCG is therefore capableof conferring multiple beneficial effects at the molecular level,including the rapid consumption of glucose and the attenuation ofcholesterol.

Methods of Treating and Preventing Immunological, Neurological, andMetabolic Conditions

In addition to methods of modulating serum lipid levels, the inventionprovides methods of treating a variety of other diseases and conditionsby administration of BCG. For instance, the methods of the invention canbe used to diagnose the presence of an immunological and/or neurologicaldisorder (e.g., by assessing the level and/or presence of one or more ofthe biomarkers described herein), and, once diagnosed, the subject canbe treated by administration of BCG. BCG induces the secretion of TNFα,a TNFR2 agonist, which activates the proliferation of regulatory T-cells(T-reg) that attenuate the growth of T- and B-lymphocytes thatcross-react with self antigens and epitopes from other non-threateningmolecules. Exemplary disorders that can be treated by administration ofBCG include autoimmune diseases, such as type I diabetes, AlopeciaAreata, Ankylosing Spondylitis, Antiphospholipid Syndrome, AutoimmuneAddison's Disease, Autoimmune Hemolytic Anemia, Autoimmune Hepatitis,Behcet's Disease, Bullous Pemphigoid, Cardiomyopathy, CeliacSprue-Dermatitis, Chronic Fatigue Immune Dysfunction Syndrome (CFIDS),Chronic Inflammatory Demyelinating Polyneuropathy, Churg-StraussSyndrome, Cicatricial Pemphigoid, CREST Syndrome, Cold AgglutininDisease, Crohn's Disease, Essential Mixed Cryoglobulinemia,Fibromyalgia-Fibromyositis, Graves' Disease, Guillain-Barré, Hashimoto'sThyroiditis, Hypothyroidism, Idiopathic Pulmonary Fibrosis, IdiopathicThrombocytopenia Purpura (ITP), IgA Nephropathy, Juvenile Arthritis,Lichen Planus, Lupus, Ménière's Disease, Mixed Connective TissueDisease, Multiple Sclerosis, Myasthenia Gravis, Pemphigus Vulgaris,Pernicious Anemia, Polyarteritis Nodosa, Polychondritis, PolyglandularSyndromes, Polymyalgia Rheumatica, Polymyositis and Dermatomyositis,Primary Agammaglobulinemia, Primary Biliary Cirrhosis, Psoriasis,Raynaud's Phenomenon, Reiter's Syndrome, Rheumatic Fever, RheumatoidArthritis, Sarcoidosis, Scleroderma, Sjögren's Syndrome, Stiff-ManSyndrome, Takayasu Arteritis, Temporal Arteritis/Giant Cell Arteritis,Ulcerative Colitis, Uveitis, Vasculitis, Vitiligo, and Wegener'sGranulomatosis. In some embodiments of the invention, subjects that aretreated for any of these autoimmune diseases are administered BCG andare not administered a TNFR2 agonist.

Other diseases that can be treated by administration of BCG includeneurological conditions, such as a brain tumor, a brain metastasis, aspinal cord injury, schizophrenia, epilepsy, Amyotrophic lateralsclerosis (ALS), Parkinson's disease, Autism, Alzheimer's disease,Huntington's disease, and stroke. In some embodiments of the invention,subjects that are treated for any of these neurological conditions areadministered BCG and are not administered a TNFR2 agonist.

Additionally, BCG can be administered to a subject in order to treat anallergy, such as a food allergy, seasonal allergy, pet allergy, hives,hay fever, allergic conjunctivitis, poison ivy allergy oak allergy, moldallergy, drug allergy, dust allergy, cosmetic allergy, or chemicalallergy. In some embodiments of the invention, subjects that are treatedfor any of these allergies are administered BCG and are not administereda TNFR2 agonist.

BCG can be administered to a subject, e.g., a mammalian subject, such asa human, suffering from a graft rejection. BCG may treat graftrejections, e.g., by stimulating the production of TNFα, which in turnmay bind TNFR2 receptors on the surface of autoreactive CD8+ T-cellsthat cross-react with antigens presented on the surface of the graft andinduce apoptosis in these CD8+ T-cells, or may stimulate the expansionof T-reg cells that may subsequently eliminate autoreactive CD8+T-cells. Examples of graft rejections that can be treated byadministration of BCG include, without limitation, skin graft rejection,bone graft rejection, vascular tissue graft rejection, ligament graftrejection, or organ graft rejection. Exemplary ligament graft rejectionsthat can be treated according to the methods of the invention includecricothyroid ligament graft rejection, periodontal ligament graftrejection, suspensory ligament of the lens graft rejection, palmarradiocarpal ligament graft rejection, dorsal radiocarpal ligament graftrejection, ulnar collateral ligament graft rejection, radial collateralligament graft rejection, suspensory ligament of the breast graftrejection, anterior sacroiliac ligament graft rejection, posteriorsacroiliac ligament graft rejection, sacrotuberous ligament graftrejection, sacrospinous ligament graft rejection, inferior pubicligament graft rejection, superior pubic ligament graft rejection,anterior cruciate ligament graft rejection, lateral collateral ligamentgraft rejection, posterior cruciate ligament graft rejection, medialcollateral ligament graft rejection, cranial cruciate ligament graftrejection, caudal cruciate ligament graft rejection, and patellarligament graft rejection. Example of organ graft rejections that can betreated according to the methods of the invention include heart graftrejection, lung graft rejection, kidney graft rejection, liver graftrejection, pancreas graft rejection, intestine graft rejection, andthymus graft rejection. In some embodiments of the invention, subjectsthat are treated for any of these graft rejections are administered BCGand are not administered a TNFR2 agonist.

BCG can also be used to treat a patient in need of organ repair orregeneration, e.g., by inducing the proliferation of cells within adamaged tissue or organ. BCG can be administered to a mammalian subject,such as a human, to stimulate TNFα secretion, which may in turn bindTNFR2 on the surface of cells within damaged tissue so as to induceTRAF2/3- and/or NFκB-mediated cell proliferation. The growth of T-regcells (e.g., CD4+, CD25+, FOXP3+T-reg cells) that is induced by BCGadministration can subsequently modulate the activity of T- andB-lymphocytes that cross-react with cells of endogenous organs ortissues. For instance, the stimulation of TNFα secretion can have theeffect of reducing populations of cytotoxic T-lymphocytes (e.g., CD8+T-cells) that are often associated with mounting an inappropriate immuneresponse that can cause an immunological disorder. In certain cases, BCGmay be capable of reducing the growth of a population of CD8+ T-cells,e.g., by about 50% to about 200% relative to untreated cells (e.g., 50%,75%, 100%, 125%, 150%, 175%, or 200%). Examples of tissues and organsthat may be induced to regenerate by administration of BCG to a subject(e.g., a mammalian subject, such as a human) include the pancreas,salivary gland, pituitary gland, kidney, heart, lung, hematopoieticsystem, cranial nerves, heart, blood vessels including the aorta,olfactory gland, ear, nerves, structures of the head, eye, thymus,tongue, bone, liver, small intestine, large intestine, gut, lung, brain,skin, peripheral nervous system, central nervous system, spinal cord,breast, embryonic structures, embryos, and testes. In some embodimentsof the invention, subjects that are administered BCG to stimulate organrepair or regeneration are administered BCG and are not administered aTNFR2 agonist.

In each case, the subject may be diagnosed as having a disease bydetecting one or more of the biomarkers described herein, such asmethylated cytosine residues within a nuclear gene (e.g., FoxP3 orCD45), a cytokine (e.g., IL-6, TNFα, or IFNγ), a lipolytic protein(e.g., acyl co-enzyme A oxidase, carnitine palmitoyltransferase, lipase,or uncoupling protein), a lipogenic protein (e.g., acetyl co-enzyme Acarboxylase α, acetyl co-enzyme A carboxylase β, fatty acid synthase,glyceraldehydes-6-phosphate dehydrogenase, stearoyl-CoA saturase, malicenzyme, or glucose-6-phosphate dehydrogenase), adiponectin receptor(e.g., adiponectin receptor 1 or adiponectin receptor 2), lysineacetyltransferase (e.g., KAT2A, KAT2B, KAT5, KAT6A, KAT6B, KAT7, orKAT8), histone acetyltransferase, histone deacetylase (e.g., HDAC1,HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11,HDAC1P1, HDAC1P2, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, or SIRT7),histone (e.g., H2A, H2B, H3, or H4), an mRNA molecule encoding any ofthese proteins, an acetylated amino acid (e.g., N-acetylalanine,N-acetylaspartic acid, N-acetylserine, N-acetylthreonine,N-acetylhistidine, N-acetyl-3-methylhistidine, N-acetylvaline, andN-α-acetyllysine, and N-acetylmethionine), or a methylated metabolite(e.g., N-α-acetyl-3-methylhistidine, 3-methylglutaconic acid,3-methylglutarylcarnitine, N-ε-trimethyllysine, 4-methyl-2-oxopentanoicacid, and 3-methyl-2-oxobutyric acid).

Methods of Treating Hyperglycemia and Associated Disorders

Another basis for the present invention is the discovery that BCG iscapable of suppressing blood glucose levels in patients in need thereof(e.g., mammalian patients, such as human patients), such ashyperglycemic patients. I have discovered that BCG is capable ofinducing a metabolic conversion from a state of oxidativephosphorylation to a state of aerobic glycolysis. A result of thisconversion is the rapid consumption of glucose in vivo. This beneficialeffect of BCG is shown, for instance, in FIGS. 3, 8A, and 8B, whichdemonstrate the ability of BCG to suppress and stabilize glycatedhemoglobin levels in human subjects. Glycated hemoglobin is an indicatorof total blood glucose concentration and can be used to detecthyperglycemic subjects. BCG is capable of inducing this conversion,known as the Warburg Effect, by augmenting the expression ofhypoxia-inducible factor 1-α (HIF1-α). As shown in FIGS. 9 and 10, BCGadministration is capable of up-regulating HIF1-α and promotes theexpression of glucose transporters and early glycolytic enzymes. Asglycolysis is less thermodynamically efficient than oxidativephosphorylation, this conversion increases glucose consumptionsubstantially to meet the demand for adenosine triphosphate.Administration of BCG thus provides an innovative method to lower bloodsugar. This mechanism is unique and provides advantages over insulintreatment, as no hypoglycemia occurs due to administration of BCG andthe blood sugar lowering effect due to BCG treatment is more robust thanthat achieved with standard insulin therapy. The use of BCG thusrepresents a method for reducing blood glucose or to replace the use ofinsulin to control blood sugar, regardless of underlying etiology.

Patients that may be suitably treated (e.g., to reduce blood sugarlevel) using BCG include those suffering from a disease associated withan elevated blood glucose level, such as type 2 diabetes,noninsulin-dependent diabetes mellitus (NIDDM), nonalcoholicsteatohepatitis (NASH), metabolic syndrome, cystic fibrosis, druginduced hyperglycemia, insulin resistance syndromes, diseases caused bygenetic mutations in the pancreas, cancer, infection, Leprechaunism,Rabson Mandenhall syndrome, lipoatrophic diabetes, pancreatitis, trauma,hemochromatoisis, fibrocalculous pancreatopathy, acromegaly, Cushingssyndrome, glucagonoma, pheochromocytoma, hyperthyroism, somatostatinoma,aldosteroma, infections associated with beta cell destruction, Rubella,coxsachie virus B, mumps, cytomegatolovirus infection, adenovirusinfection, a genetic syndrome, stiff person syndrome, anti-insulinreceptor abnormalities, liver disease, and renal failure. In someembodiments, the drug induced hyperglycemia is induced by one or moreagents selected from the group consisting of steroids, cortisol,thiazides, diazocide, calcineurin inhibitors, oral contraceptives, betaadrenergic agonists, nicotinic acid, pentamidine, alpha interferon,anti-psychotic agents, anti-retroviral agents, and rodenticides (e.g.,pyrinuron). In some embodiments, the cancer is pancreatic cancer. Insome embodiments, the genetic syndrome is selected from the groupconsisting of Down's syndrome, Klinefelter's syndrome, Turner syndrome,Woldfram syndrome, and Friendreich ataxia. In some embodiments, thesubject has undergone a pancreatectomy. The subject may exhibit one ormore mutations in a mitochondrial gene, such as hepatic nuclear factor 1(MODY3), glucokinase (MODY2), and hepatocyte nuclear factor 4-α (MODY1).

Additionally or alternatively, BCG administration can alleviate orreduce a symptom associated with the disease, such as polyphagia,polydipsia, polyuria, blurred vision, fatigue, cardiac arrhythmia,stupor, dry mouth, and poor wound healing.

Methods of Combination Therapy

In order to treat a subject having one of the conditions describedherein (e.g., a subject that has already been diagnosed as having one ofthe conditions described herein), BCG may be administered to the subjectin conjunction with another therapeutic agent. For instance, a subjectthat has an elevated level of serum cholesterol, LDLs, and/ortriglycerides, and/or that is in need of an increase in serum HDLlevels, may be administered a hypolipidemic agent in addition to BCG.This can be performed by admixing BCG together with a hypolipidemicagent or by administering an effective amount of BCG separately from thehypolipidemic agent. Exemplary hypolipidemic agents for use with themethods of the invention include HMG-CoA reductase inhibitors, niacin,fibric acid derivatives, cholesterol absorption inhibitors, andlipolytic agents. For instance, a HMG-CoA reductase inhibitors that maybe admixed with or administered separately from BCG in order to treat asubject with elevated levels of cholesterol, LDLs, and/or triglycerides,and/or that is in need of an increase in the level of serum HDLs,include atorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin,pitavastatin, pravastatin, rosuvastatin, simvastatin, and combinationsthereof. Examples of fibric acid derivatives that can be admixed with oradministered separately from BCG and administered to a subject accordingto the methods of the invention include fenofibrate and gemfibrozil.Additionally, ezetimibe((3R,4S)-1-(4-fluorophenyl)-3-[(3S)-3-(4-fluorophenyl)-3-hydroxypropyl]-4-(4-hydroxyphenyl)azetidin-2-one)is a fibric acid derivative that can be admixed with or administeredseparately from BCG to modulate serum lipid levels (e.g., to reduceserum cholesterol, LDL, and/or triglyceride levels, and/or to increaseserum HDL levels) in a subject according to the methods of theinvention. Exemplary lipolytic agents that can be admixed with oradministered separately from BCG to modulate serum lipid levels in asubject include norepinephrine, isoproterenol, forskolin, bucladesine,and theophylline.

In addition to agents that directly regulate lipid metabolism, otheragents that can synergize with BCG in the treatment of a subject havingelevated serum cholesterol, LDL, and/or triglyceride levels, and/or thatis in need of an increase in serum HDL levels, include TNFR2 agonists,such as TNFα. These agents are capable of potentiating the proliferationof various cells, such as T-reg cells, and can be administered inconjunction with BCG to a subject in order to reduce serum cholesterol,LDLs, and/or triglycerides, and/or to elevate serum HDLs. Additionalagents that can be admixed, conjugated, or administered with, oradministered separately from BCG to stimulate TNFR2 activity includee.g., IL-2, TNFα, as well as agonistic TNFR2 antibodies (see, e.g., WO2014/124134, the disclosure of which is incorporated herein byreference).

Additionally, BCG may be admixed with or administered separately from animmunotherapy agent in order to reduce serum cholesterol, LDLs, and/ortriglycerides, and/or to elevate serum HDLs. Exemplary immunotherapyagents include antibodies, fragments thereof, and other proteins capableof binding a particular epitope or target molecule. Exemplaryimmunotherapy agents useful in conjunction with the compositions andmethods of the invention include an anti-CTLA-4 agent, an anti-PD-1agent, an anti-PD-L1 agent, an anti-PD-L2 agent, a TNFα cross-linkingagent, a TRAIL cross-linking agent, a CD27 agent, a CD30 agent, a CD40agent, a 4-1 BB agent, a GITR agent, an OX40 agent, a TRAILR1 agent, aTRAILR2 agent, a TWEAKR agent, and, e.g., agents directed toward theimmunological targets described in Table 1 of Mahoney et al., CancerImmunotherapy, 14:561-584 (2015), the disclosure of which isincorporated herein by reference. For example, immunological target 4-1BB ligand may be targeted with an anti-4-1 BB ligand antibody;immunological target OX40L may be targeted with an anti-OX40L antibody;immunological target GITR may be targeted with an anti-GITR antibody;immunological target CD27 may be targeted with an anti-CD27 antibody;immunological target TL1A may be targeted with an anti-TL1A antibody;immunological target CD40L may be targeted with an anti-CD40L antibody;immunological target LIGHT may be targeted with an anti-LIGHT antibody;immunological target BTLA may be targeted with an anti-BTLA antibody;immunological target LAG3 may be targeted with an anti-LAG3 antibody;immunological target TIM3 may be targeted with an anti-TIM3 antibody;immunological target Singlecs may be targeted with an anti-Singlecsantibody; immunological target ICOS ligand may be targeted with ananti-ICOS ligand antibody; immunological target B7-H3 may be targetedwith an anti-B7-H3 antibody; immunological target B7-H4 may be targetedwith an anti-B7-H4 antibody; immunological target VISTA may be targetedwith an anti-VISTA antibody; immunological target TMIGD2 may be targetedwith an anti-TMIGD2 antibody; immunological target BTNL2 may be targetedwith an anti-BTNL2 antibody; immunological target CD48 may be targetedwith an anti-CD48 antibody; immunological target KIR may be targetedwith an anti-KIR antibody; immunological target LIR may be targeted withan anti-LIR antibody; immunological target ILT may be targeted with ananti-ILT antibody; immunological target NKG2D may be targeted with ananti-NKG2D antibody; immunological target NKG2A may be targeted with ananti-NKG2A antibody; immunological target MICA may be targeted with ananti-MICA antibody; immunological target MICB may be targeted with ananti-MICB antibody; immunological target CD244 may be targeted with ananti-CD244 antibody; immunological target CSF1R may be targeted with ananti-CSF1R antibody; immunological target IDO may be targeted with ananti-IDO antibody; immunological target TGFβ may be targeted with ananti-TGFβ antibody; immunological target CD39 may be targeted with ananti-CD39 antibody; immunological target CD73 may be targeted with ananti-CD73 antibody; immunological target CXCR4 may be targeted with ananti-CXCR4 antibody; immunological target CXCL12 may be targeted with ananti-CXCL12 antibody; immunological target SIRPA may be targeted with ananti-SIRPA antibody; immunological target CD47 may be targeted with ananti-CD47 antibody; immunological target VEGF may be targeted with ananti-VEGF antibody; and immunological target neuropilin may be targetedwith an anti-neuropilin antibody (see, e.g., Table 1 of Mahoney et al.)Immunotherapy agents described herein may be expressed recombinantly,e.g., from cultured cells (such as mammalian cells, e.g., CHO cells orHEK293 cells), or bacterial cells, such as E. coli.

Immunotherapy agents can be expressed using standard recombinant DNAtechniques known in the art, e.g., by transfecting a population ofcultured cells with a plasmid containing one or more genes encoding animmunotherapy agent operably linked to a regulatory sequence known inthe art, such as a promoter or enhancer capable of directingtranscription of the gene of interest. Suitable vectors for thetransfection of eukaryotic and prokaryotic cells are known in the art.Immunotherapy agents may optionally be produced as fusion proteinscontaining more than one therapeutic or biologically active moietychemically bound together, e.g., by a linker known in the art ordescribed herein.

Pharmaceutical Compositions

Therapeutic compositions containing BCG can be prepared, e.g., usingmethods known in the art or described herein. For instance, BCGformulations can be prepared using physiologically acceptable carriers,excipients, and/or stabilizers (Remington's Pharmaceutical Sciences 16thedition, Osol, A. Ed. (1980); the disclosure of which is incorporatedherein by reference), and in a desired form, e.g., in the form ofaqueous solutions or suspensions. The compositions can also be preparedso as to contain BCG at a desired concentration or cell count. BCGcompositions of the invention also include lyophilized compositions thatcan be rehydrated prior to administration. The sections that followdescribe useful additives that can be included in a BCG formulation foradministration to a subject or for long-term storage.

Cryopreserved Formulations of BCG

Pharmaceutical compositions of BCG can be prepared for storage bycryopreservation, e.g., by contacting BCG with a cryoprotectant known inthe art, such as dimethylsulfoxide (DMSO). Suitable DMSO concentrationsin BCG stock solutions range from 0.01% to about 1% DMSO. Cryopreservedsolutions can include acceptable carriers, excipients or stabilizerstypically employed in the art, e.g., buffering agents, stabilizingagents, preservatives, isotonifiers, non-ionic detergents, antioxidants,and other miscellaneous additives. See, e.g., Remington's PharmaceuticalSciences, 16th edition (Osol, ed. 1980; incorporated herein byreference). Such additives are generally nontoxic to the subject that isultimately treated at the dosages and concentrations employed.

Buffering Agents

A wide array of buffering agents can be included in a BCG formulationuseful in conjunction with the methods of the invention. Thesesubstances serve to maintain the pH of the formulation in a desirablerange, e.g., a range that approximates physiological conditions.Buffering agents can be present at concentration ranging from, e.g.,about 2 mM to about 50 mM. Suitable buffering agents for use with BCGformulations include both organic and inorganic acids and salts thereofsuch as citrate buffers (e.g., monosodium citrate—disodium citratemixture, citric acid—trisodium citrate mixture, citric acid—monosodiumcitrate mixture, etc.), succinate buffers (e.g., succinicacid—monosodium succinate mixture, succinic acid—sodium hydroxidemixture, succinic acid—disodium succinate mixture, etc.), tartratebuffers (e.g., tartaric acid—sodium tartrate mixture, tartaricacid—potassium tartrate mixture, tartaric acid—sodium hydroxide mixture,etc.), fumarate buffers (e.g., fumaric acid—monosodium fumarate mixture,fumaric acid—disodium fumarate mixture, monosodium fumarate—disodiumfumarate mixture, etc.), gluconate buffers (e.g., gluconic acid—sodiumglyconate mixture, gluconic acid—sodium hydroxide mixture, gluconicacid—potassium glyuconate mixture, etc.), oxalate buffer (e.g., oxalicacid—sodium oxalate mixture, oxalic acid—sodium hydroxide mixture,oxalic acid—potassium oxalate mixture, etc.), lactate buffers (e.g.,lactic acid—sodium lactate mixture, lactic acid—sodium hydroxidemixture, lactic acid—potassium lactate mixture, etc.) and acetatebuffers (e.g., acetic acid—sodium acetate mixture, acetic acid—sodiumhydroxide mixture, etc.). Additionally, phosphate buffers, histidinebuffers and trimethylamine salts such as Tris can be used.

Preservatives

Preservatives can be added to a formulation of BCG and, optionally, anadditional therapeutic agent, in order to retard the growth of otherpotential microbes in the pharmaceutical composition. For instance,preservatives can be present in a BCG-containing formulation in a widerange of concentrations, e.g., ranging from 0.02%-1% (w/v). Suitablepreservatives for use in a pharmaceutical composition of the inventioninclude phenol, benzyl alcohol, meta-cresol, methyl paraben, propylparaben, octadecyldimethylbenzyl ammonium chloride, benzalconium halides{e.g., chloride, bromide, and iodide), hexamethonium chloride, and alkylparabens such as methyl or propyl paraben, catechol, resorcinol,cyclohexanol, and 3-pentanol. Isotonicifiers can be added to ensureisotonicity of BCG formulations and include polhydric sugar alcohols,for example trihydric or higher sugar alcohols, such as glycerin,arabitol, xylitol, sorbitol and mannitol.

BCG formulations useful in conjunction with the methods of the inventionmay include stabilizers. Stabilizers represent a broad category ofexcipients which can range in function from a bulking agent to anadditive which solubilizes an additional therapeutic agent or helps toprevent denaturation or adherence of the therapeutic agent to thecontainer wall. Typical stabilizers can be polyhydric sugar alcohols(enumerated above); amino acids such as arginine, lysine, glycine,glutamine, asparagine, histidine, alanine, ornithine, L-leucine,2-phenylalanine, glutamic acid, threonine, etc., organic sugars or sugaralcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol,xylitol, ribitol, myoinisitol, galactitol, glycerol and the like,including cyclitols such as inositol; polyethylene glycol; amino acidpolymers; sulfur containing reducing agents, such as urea, glutathione,thioctic acid, sodium thioglycolate, thioglycerol, a-monothioglyceroland sodium thio sulfate; low molecular weight polypeptides (e.g.,peptides of 10 residues or fewer); proteins such as human serum albumin,bovine serum albumin, gelatin or immunoglobulins; hydrophylic polymers,such as polyvinylpyrrolidone monosaccharides, such as xylose, mannose,fructose, glucose; disaccharides such as lactose, maltose, sucrose andtrisaccharides such as raffinose; and polysaccharides such as dextran.Stabilizers can be present in a BCG formulation in a wide range ofconcentrations, e.g., from 0.001% to 10.0% (w/w).

Detergents

Non-ionic surfactants or detergents (also known as “wetting agents”) canbe added to help solubilize the therapeutic agent (e.g., an additionaltherapeutic agent co-formulated with BCG) as well as to protect thetherapeutic agent against agitation-induced aggregation, which alsopermits the formulation to be exposed to shear surface stressed withoutcausing denaturation of a therapeutic protein (e.g., an immunotherapyagent). Suitable non-ionic surfactants that can be added to aformulation containing BCG and, optionally, an additional therapeuticagent include polysorbates (20, 80, etc.), polyoxamers (184, 188 etc.),Pluronic polyols, polyoxyethylene sorbitan monoethers (TWEEN®-20,TWEEN®-80, etc.). Non-ionic surfactants can be present in a range ofabout 0.05 mg/mL to about 1.0 mg/mL, for example about 0.07 mg/mL toabout 0.2 mg/mL. Additional miscellaneous excipients that can be addedto a formulation containing BCG and, optionally, an additionaltherapeutic agent include bulking agents (e.g., starch), chelatingagents (e.g., EDTA), antioxidants (e.g., ascorbic acid, methionine,vitamin E), and cosolvents.

Other Pharmaceutical Carriers

Alternative pharmaceutically acceptable carriers that can beincorporated into a BCG formulation may include dextrose, sucrose,sorbitol, mannitol, starch, rubber arable, potassium phosphate,arginate, gelatin, potassium silicate, microcrystalline cellulose,polyvinylpyrrolidone, cellulose, water, syrups, methyl cellulose,methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesiumstearate, and mineral oils. A composition containing BCG may furtherinclude a lubricant, an emulsifier, a suspending agent, and apreservative. Details of suitable pharmaceutically acceptable carriersand formulations can be found in Remington's Pharmaceutical Sciences(19th ed., 1995), which is incorporated herein by reference.

Blood-Brain Barrier Penetration

In certain embodiments, therapeutic agents described herein can beformulated to ensure proper distribution in vivo. For example, theblood-brain barrier (BBB) excludes many high-molecular weight compounds,as well as those with elevated hydrophilicity. To ensure that thetherapeutic agents useful with the methods of the invention cross theBBB (if desired), they can be formulated, for example, in liposomes.Methods of manufacturing liposomes have been described, e.g., U.S. Pat.Nos. 4,522,811; 5,374,548; and 5,399,331. The liposomes may comprise oneor more moieties that are selectively transported into specific cells ororgans, thereby enhancing targeted drug delivery (see, e.g., V. V.Ranade, J. Clin. Pharmacol. 29:685, 1989)). Exemplary targeting moietiesinclude, e.g., folate or biotin (see, e.g., U.S. Pat. No. 5,416,016);mannosides (Umezawa et al. (Biochem. Biophys. Res. Commun. 153:1038,1988)); antibodies (P. G. Bloeman et al. (FEBS Lett. 357:140, 1995); M.Owais et al. (Antimicrob. Agents Chemother. 39:180, 1995)); andsurfactant protein A receptor (Briscoe et al. (Am. J. Physiol. 1233:134,1995)); the disclosures of each of which are incorporated herein byreference.

Routes of Administration and Dosing Unit Dosage Forms

Desirably, the methods of the invention include administering BCG to asubject in a unit dosage form that contains a low quantity of BCG thatis capable of inducing a therapeutic response (e.g., lowering serumcholesterol, LDL, and/or triglyceride levels, increasing serum HDLlevels, and/or lowering blood glucose levels). A BCG formulation usefulin conjunction with the methods of the invention may contain, e.g.,between 1×10⁴ and 1×10⁹ cfu per 0.1 milligrams of BCG (e.g., 1×10⁴,2×10⁴, 3×10⁴, 4×10⁴, 5×10⁴, 6×10⁴, 7×10⁴, 8×10⁴, 9×10⁴, 1×10⁵, 2×10⁵,3×10⁵, 4×10⁵, 5×10⁵, 6×10⁵, 7×10⁵, 8×10⁵, 9×10⁵, 1×10⁶, 2×10⁶, 3×10⁶,4×10⁶, 5×10⁶, 6×10⁶, 7×10⁶, 8×10⁶, 9×10⁶, 1×10⁷, 2×10⁷, 3×10⁷, 4×10⁷,5×10⁷, 6×10⁷, 7×10⁷, 8×10⁷, 9×10⁷, 1×10⁸, 2×10⁸, 3×10⁸, 8×10⁸, 5×10⁸,6×10⁸, 7×10⁸, 8×10⁸, 9×10⁸, or 1×10⁹ cfu per 0.1 milligrams of BCG.

In preferred embodiments, BCG is administered to a subject in a unitdosage form containing between about 5×10⁵ and about 1×10⁷ cfu per 0.1milligrams of BCG. For instance, preferred unit dosage forms of BCGuseful in conjunction with the methods of the invention contain 5×10⁵,6×10⁵, 7×10⁵, 8×10⁵, 9×10⁵, 1×10⁶, 2×10⁶, 3×10⁶, 4×10⁶, 5×10⁶, 6×10⁶,7×10⁶, 8×10⁶, 9×10⁶, or 1×10⁷ cfu per 0.1 milligrams of BCG. Inparticularly preferred embodiments, BCG is administered to a subject ina unit dosage form containing between about 1×10⁶ and 6×10⁶ cfu per 0.1milligrams of BCG. Desirably, unit dosage forms useful with the methodsof the invention may contain about 3.9×10⁶ cfu per 0.1 milligrams ofBCG.

BCG can be used in conjunction with the compositions and methods of theinvention, as well as any other form of mycobacterium. Exemplarymycobacteria useful in conjunction with the compositions and methods ofthe invention include substrains of BCG. Substrains of BCG include thosethat can be cultured under good manufacturing protocols, such as thePasteur, Japan-Tokyo, Pasteur, Copenhagen, TICE, Sanofi, Connaught,RIVM, Evans, MMC, and Glaxo substrains of BCG, as well as attenuated andgenetically modified versions of these strains. Mycobacteria for usewith the compositions and methods of the invention may be live,attenuated, or inactivated such that the bacteria retain certain antigenexpression patterns but are no longer virulent.

Re-Dosing Based on Diagnostic Methods of the Invention

In various cases, it may be desirable to administer BCG to a subjectmultiple times (e.g., between 2 and 20 times, such as 2, 3, 4, 5, 6, 7,8, 9, or 10 times) based on the response of a subject to BCG therapy.For instance, a subject may be administered BCG according to the methodsof the invention in order to reduce serum cholesterol levels or to treatone or more of the diseases described herein. After a period of timefollowing this initial administration (e.g., one or more days, weeks,months, or years), the subject may be re-evaluated in order to determinethe continued effectiveness of the initial BCG administration on thedisease or conditions being treated. For example, a subject that isadministered BCG or another therapeutic agent in order to reduce serumcholesterol levels may be examined using one or more analytical tests(such as those described herein) in order to assess the responsivenessof the subject to administration of BCG or another therapeutic agentand/or to determine if the subject would benefit from additional dosesof BCG or another medicament. The subject may be administered BCG oranother therapeutic agent and may subsequently have a blood samplewithdrawn in order to determine if the methylation state of one or morecytosine residues in the nuclear DNA within a cell of the subject haschanged in response to administration of BCG or another therapeuticagent. For instance, a physician of skill in the art can withdraw ablood sample from a subject having previously been administered BCG oranother therapeutic agent and may analyze the nuclear DNA within a cellof the sample (e.g., within a T-lymphocyte of the sample, such as aCD4+, CD25+T-reg cell) to determine if one or more cytosine residues hasbeen methylated or demethylated in response to treatment with BCG oranother therapeutic agent. Preferably, the one or more cytosine residuesare located within a gene that encodes a transcription factor, such asFoxP3, or within a gene that encodes CD45. A determination that thequantity of methylated cytosine residues in one or more of both of thesegenetic loci has decreased indicates that the patient is responding totreatment with BCG or another therapeutic agent and may not requiresubsequent doses. For instance, a determination that one or moreparticular cytosine residues within the FoxP3 and/or CD45 genes that waspreviously methylated in the subject prior to treatment with BCG oranother therapeutic agent treatment and has since undergonedemethylation indicates that the subject is responding to the therapyand may not require subsequent doses of BCG or another medicament.

In contrast, a determination that the quantity of methylated cytosineresidues in one or both of these genetic loci has stayed the same orincreased indicates that the subject would likely benefit from one ormore additional doses of BCG or another therapeutic agent. For example,a determination that one or more particular cytosine residues within theFoxP3 and/or CD45 genes that was previously methylated in the subjectprior to treatment with BCG or another therapeutic agent and remainsmethylated following administration of BCG or another therapeutic agentindicates that the subject would likely benefit from one or moresubsequent doses of BCG or another therapeutic agent. Alternatively, adetermination that that one or more particular cytosine residues withinthe FoxP3 and/or CD45 genes that was previously demethylated in thesubject prior to administration of BCG or another therapeutic agent andhas since undergone methylation following administration of BCG oranother therapeutic agent indicates that the subject would likelybenefit from one or more subsequent doses of BCG or another therapeuticagent.

In addition to analysis of cytosine methylation state, one of skill inthe art may examine the level of one or more mRNA molecules, proteins,acetylated amino acids, and/or methylated metabolites in a sampleisolated from a subject having previously been administered BCG oranother therapeutic agent in order to determine if the subject isresponding to the therapy or if he/she would benefit from receivingadditional doses of BCG or another medicament. For instance, accordingto the methods of the invention, one of skill in the art may determinethe level of one or more mRNA molecules encoding a cytokine or lipolyticprotein in order to assess whether a subject previously administered BCGor another therapeutic agent would likely benefit from receivingadditional doses. A determination that the level of one or more mRNAmolecules encoding a cytokine (e.g., IL-6, TNFα, or IFNγ) or a lipolyticprotein (e.g., acyl co-enzyme A oxidase, carnitine palmitoyltransferase,lipase, or uncoupling protein) in a sample isolated from a subject thathas been previously administered BCG or another therapeutic agent isabout the same as or is less than the level of the same mRNA moleculesin a sample previously isolated from the subject (e.g., between about 24hours and about 5 years prior to the subject having been administeredBCG or another therapeutic agent, preferably between about 24 hours andabout 5 years prior to administration of BCG or the other therapeuticagent, and desirably between about 1 month and 1 year prior to thesubject having been administered BCG or the other therapeutic agent)indicates that the subject would likely benefit from receiving one ormore additional doses of BCG or another therapeutic agent.

Alternatively, one of skill in the art may determine the level of one ormore mRNA molecules encoding a lipogenic protein, an adiponectinreceptor, a lysine acetyltransferase (KAT), a histone acetyltransferase,a histone deacetylase (HDAC), or a histone in order to assess whether asubject previously administered BCG or another therapeutic agent wouldlikely benefit from receiving additional doses of BCG or the previouslyadministered therapeutic agent (in particular, BCG). A determinationthat the level of one or more mRNA molecules encoding a lipogenicprotein (e.g., acetyl co-enzyme A carboxylase α, acetyl co-enzyme Acarboxylase β, fatty acid synthase, glyceraldehydes-6-phosphatedehydrogenase, stearoyl-CoA saturase, malic enzyme, orglucose-6-phosphate dehydrogenase), adiponectin receptor (e.g.,adiponectin receptor 1 or adiponectin receptor 2), lysineacetyltransferase (e.g., KAT2A, KAT2B, KAT5, KAT6A, KAT6B, KAT7, orKAT8), histone acetyltransferase, histone deacetylase (e.g., HDAC1,HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11,HDAC1P1, HDAC1P2, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, or SIRT7),or histone (e.g., H2A, H2B, H3, or H4) in a sample isolated from asubject that has been previously administered BCG or another therapeuticagent is about the same as or is greater than the level of the same mRNAmolecules in a sample previously isolated from the subject (e.g.,between about 24 hours and about 5 years prior to the subject havingbeen administered BCG or another therapeutic agent, preferably betweenabout 24 hours and about 5 years prior to administration of BCG or theother therapeutic agent, and desirably between about 1 month and 1 yearprior to the subject having been administered BCG or the othertherapeutic agent) indicates that the subject would likely benefit fromreceiving one or more additional doses of BCG or another therapeuticagent (preferably BCG).

The levels of mRNA molecules in a sample isolated from a subject (e.g.,from one or more cells, such as T-lymphocytes, isolated from a subject)can be determined using methods known in the art, such as by Northernblot analysis or by standard quantitative reverse-transcriptionpolymerase chain reaction (PCR) techniques.

Additionally or alternatively, one of skill in the art may determine thelevel of one or more proteins, such as a cytokine or lipolytic protein,in order to assess whether a subject previously administered BCG oranother therapeutic agent would likely benefit from receiving additionaldoses. A determination that the level of the one or more cytokines(e.g., IL-6, TNFα, or IFNγ) or lipolytic proteins (e.g., acyl co-enzymeA oxidase, carnitine palmitoyltransferase, lipase, or uncouplingprotein) in a sample isolated from a subject that has been previouslyadministered BCG or another therapeutic agent is about the same as or isless than the level of the same proteins in a sample previously isolatedfrom the subject (e.g., between about 24 hours and about 5 years priorto the subject having been administered BCG or another therapeuticagent, preferably between about 24 hours and about 5 years prior to BCGor another therapeutic agent administration, and desirably between about1 month and 1 year prior to the subject having been administered BCG oranother therapeutic agent) indicates that the subject would likelybenefit from receiving one or more additional doses of BCG or anothertherapeutic agent.

Alternatively, one of skill in the art may determine the level of one ormore different proteins, such as a lipogenic protein, an adiponectinreceptor, a lysine acetyltransferase (KAT), a histone acetyltransferase,a histone deacetylase (HDAC), or a histone in order to assess whether asubject previously administered BCG or another therapeutic agent wouldlikely benefit from receiving additional doses of BCG or the previouslyadministered therapeutic agent (in particular, BCG). A determinationthat the level of one or more lipogenic proteins (e.g., acetyl co-enzymeA carboxylase α, acetyl co-enzyme A carboxylase β, fatty acid synthase,glyceraldehydes-6-phosphate dehydrogenase, stearoyl-CoA saturase, malicenzyme, or glucose-6-phosphate dehydrogenase), adiponectin receptors(e.g., adiponectin receptor 1 or adiponectin receptor 2), lysineacetyltransferases (e.g., KAT2A, KAT2B, KAT5, KAT6A, KAT6B, KAT7, orKAT8), histone acetyltransferases, histone deacetylases (e.g., HDAC1,HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11,HDAC1P1, HDAC1P2, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, or SIRT7),or histones (e.g., H2A, H2B, H3, or H4) in a sample isolated from asubject that has been previously administered BCG or another therapeuticagent is about the same as or is greater than the level of the sameproteins a sample previously isolated from the subject (e.g., betweenabout 24 hours and about 5 years prior to the subject having beenadministered BCG or another therapeutic agent, preferably between about24 hours and about 5 years prior to treatment with BCG or anothertherapeutic agent, and desirably between about 1 month and 1 year priorto the subject having been administered BCG or the other therapeuticagent) indicates that the subject would likely benefit from receivingone or more additional doses of BCG or another therapeutic agent(preferably BCG).

The levels of the proteins described herein can be monitored usingstandard techniques know in the art, such as by solution phase ELISAassays or by immunoblot assays, such as Western blot experiments knownin the field.

In addition to monitoring mRNA and protein levels to assess thelikelihood that a subject would benefit from one or more subsequentdoses of BCG or another therapeutic agent, one of skill in the art canalso monitor the level of one or more acetylated amino acids ormethylated metabolites in order to make this determination. Forinstance, one of skill in the art can monitor the level of one or moreacetylated amino acids, such as n-acetylalanine, N-acetylaspartic acid,N-acetylserine, N-acetylthreonine, N-acetylhistidine,N-acetyl-3-methylhistidine, N-acetylvaline, and N-α-acetyllysine, orN-acetylmethionine, in a sample isolated from a subject that haspreviously been administered BCG or another therapeutic agent.Additionally or alternatively, one of skill in the art may determine thelevel of one or more methylated metabolites, such asN-α-acetyl-3-methylhistidine, 3-methylglutaconic acid,3-methylglutarylcarnitine, or N-ε-trimethyllysine, in a sample isolatedfrom the subject. A determination that the level of one or more of theseacetylated amino acids and/or methylated metabolites in the sampleisolated from the subject is about the same as or less than the levelsof the same acetylated amino acids and/or methylated metabolites in asample previously isolated from the subject (e.g., between about 24hours and about 5 years prior to the subject having been administeredBCG or another therapeutic agent, preferably between about 24 hours andabout 5 years prior to BCG or another therapeutic agent administration,and desirably between about 1 month and 1 year prior to the subjecthaving been administered BCG or the other therapeutic agent) indicatesthat the subject would likely benefit from receiving one or moreadditional doses of BCG or another therapeutic agent.

Additionally or alternatively, one of skill in the art can monitor thelevel of one or more different methylated metabolites in order to assessthe likelihood of the subject to benefit from additional doses of BCG oranother therapeutic agent. For instance, one of skill in the art canmonitor the level of one or both of 4-methyl-2-oxopentanoic acid and3-methyl-2-oxobutyric acid in a sample isolated from a subject that haspreviously been administered BCG or another therapeutic agent. Adetermination that the level of one or more of these methylatedmetabolites in the sample isolated from the subject is about the same asor less than the levels of the same methylated metabolites in a samplepreviously isolated from the subject (e.g., between about 24 hours andabout 5 years prior to the subject having been administered BCG oranother therapeutic agent, preferably between about 24 hours and about 5years prior to treatment with BCG or another therapeutic agentadministration, and desirably between about 1 month and 1 year prior tothe subject having been administered BCG or the other therapeutic agent)indicates that the subject would likely benefit from receiving one ormore additional doses of BCG or another therapeutic agent.

The levels of acetylated amino acids and methylated metabolitesdescribed herein can be determined quantitatively using establishedtechniques known in the art, including by the use of nuclear magneticresonance (NMR), high-performance liquid chromatography (HPLC),mass-spectrometry (MS), UV-Vis spectroscopy, among others.

Upon determining that a subject would likely benefit from one or moreadditional doses of BCG or another therapeutic agent, a physician ofskill in the art can administer treatment to the subject, e.g., one ormore doses of BCG. Using the methods of the invention, a physician mayadminister, e.g., between one and 20 total doses of BCG, or more, to thesubject over the course of treatment. For instance, a physician mayadminister an initial dose of BCG to the subject and, following thegenomic, proteomic, or metabolomic analyses described herein, thesubject may subsequently be administered one or more additional doses ofBCG a certain time after receiving the initial dose. The time thatpasses between the initial dose and any subsequent doses may varydepending on the patient and the conditions being treated. For example,a patient may receive an initial dose of BCG and, after a physicianassesses the subject using, e.g., one or more of the diagnosticbiomarkers described herein, and concludes that the subject would likelybenefit from one or more subsequent doses of BCG, the subject may thenbe administered additional doses of BCG, e.g., between about 1 week andabout 20 years following the initial dose. For instance, a patient maybe administered one or more doses of BCG about once every 1-10 years,such as about once every 5 years. In preferred embodiments, the subjectreceives a total of two doses of BCG: an initial dose and a follow-ondose after a physician makes the determination that the subject wouldlikely benefit from the additional administration.

As an alternative to receiving another dose of BCG, the subject caninstead be administered another therapeutic agent, depending on thedisease being treated. If the patient is being treated for elevatedserum cholesterol, LDL, or triglyceride levels, and/or for reduced HDLlevels, the patient may be administered, e.g., a hypolipidemic agent,such as those described herein or known in the art. If the patient isbeing treated for an immunological, neurological, or metabolic disorder(e.g., an autoimmune disease, a neurological condition, an allergy,allograft rejection, graft-versus-host disease, asthma, maculardegeneration, muscular atrophy, a disease related to miscarriage,atherosclerosis, bone loss, a musculoskeletal disease, and obesity), thepatient may be administered a TNFR2 agonist as a subsequent therapeuticagent, e.g., in order to stimulate the proliferation of different cellpopulations, such as T-reg cells. Additionally or alternatively, apatient being treated for one or more of these conditions may receive asa subsequent medicament an immunotherapy agent, such as an anti-CTLA-4agent, an anti-PD-1 agent, an anti-PD-L1 agent, an anti-PD-L2 agent, aTNFα cross-linking agent, a TRAIL cross-linking agent, a CD27 agent, aCD30 agent, a CD40 agent, a 4-1 BB agent, a GITR agent, an OX40 agent, aTRAILR1 agent, a TRAILR2 agent, or a TWEAKR agent.

Routes of Administration

According to the methods of the invention, BCG can be administered to asubject (e.g., a mammalian subject, such as a human) by a variety ofroutes. Using the methods of the invention, BCG is preferablyadministered to a subject intradermally, subcutaneously, orally,transdermally, intranasally, intravenously, intramuscularly,intraocularly, parenterally, intrathecally, or intracerebroventricularly(e.g., intradermally or subcutaneously). In some embodiments, BCG is notadministered to the subject (e.g., a human subject) intravenously. Themost suitable route for administration in any given case will depend onthe nature and severity of the particular disease being treated, thepatient, pharmaceutical formulation methods, the patient's age, bodyweight, sex, the patient's diet, and the patient's excretion rate.

Therapeutic formulations of BCG can be administered with medical devicesknown in the art. For example, a therapeutic formulation of BCG can beadministered with a needleless hypodermic injection device, such as thedevices disclosed in U.S. Pat. Nos. 5,399,163; 5,383,851; 5,312,335;5,064,413; 4,941,880; 4,790,824; or U.S. Pat. No. 4,596,556; thedisclosures of each of which are incorporated herein by reference.Examples of well-known implants and modules useful in the inventioninclude those described in U.S. Pat. No. 4,487,603; which discloses animplantable micro-infusion pump for dispensing medication at acontrolled rate; U.S. Pat. No. 4,486,194; which discloses a therapeuticdevice for administering medicaments through the skin; U.S. Pat. No.4,447,233; which discloses a medication infusion pump for deliveringmedication at a precise infusion rate; U.S. Pat. No. 4,447,224; whichdiscloses a variable flow implantable infusion apparatus for continuousdrug delivery; U.S. Pat. No. 4,439,196; which discloses an osmotic drugdelivery system having multi-chamber compartments; and U.S. Pat. No.4,475,196; which discloses an osmotic drug delivery system. Thesepatents are incorporated herein by reference. Many other such implants,delivery systems, and modules are known to those skilled in the art.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a description of how the compositions and methodsdescribed herein may be used, made, and evaluated, and are intended tobe purely exemplary of the invention and are not intended to limit thescope of what the inventor regards as her invention.

Example 1. Administration of BCG Promotes Sustained Decrease in SerumLipids

BCG can be administered to a subject, such as a subject (e.g., a human)that has previously been diagnosed as having elevated levels of serumcholesterol, LDLs, and/or triglycerides in order to reduce theconcentrations of one or more of these lipids and restore healthy lipidmetabolism in the subject. As shown in FIG. 1, administration of BCG toa group of subjects with elevated cholesterol promoted a decrease inserum cholesterol relative to subjects treated with a placebo instead ofBCG. Moreover, the data in FIG. 1 demonstrates that the decrease inserum cholesterol was sustained over the course of up to 7 yearsfollowing administration of BCG. Likewise, the data shown in FIG. 2demonstrate that BCG is similarly capable of attenuating serum LDLlevels over a prolonged period of time. FIG. 3 shows that BCG isadditionally capable of reducing serum levels of glycated hemoglobin,which serves as an indicator of serum glucose concentration and canoften signal an aberration in glucose metabolism. Taken together, thedata presented in FIGS. 1-3 demonstrate that BCG induces a prolongedattenuation in serum cholesterol, LDL, and HbA1c levels and represents arobust therapeutic regimen for treating these indications and diseasesassociated with elevated levels of these substances.

Example 2. Administration of BCG Restores Healthy Levels of N-AcetylatedAmino Acids and Methylated Metabolites

Administration of BCG to a subject suffering from elevated serumcholesterol, LDLs, and/or triglycerides, reduced levels of serum HDLs,and/or an immunological, neurological, or metabolic disease describedherein is capable of regulating the levels of various acetylated aminoacids and methylated metabolites. As shown in FIGS. 4A and 4B, a subjectpresenting with one or more of these diseases (e.g., type I diabetes)can be treated with BCG in order to increase the level of variousN-acetylated amino acids, such as N-acetylalanine, N-acetylasparticacid, N-acetylserine, N-acetylthreonine, N-acetylhistidine,N-acetyl-3-methylhistidine, N-acetylvaline, and N-α-acetyllysine, andN-acetylmethionine. BCG may promote an increase in one or more of thesesubstances by inducing the acetylation of, e.g., alanine, aspartic acid,serine, threonine, histidine, 3-methylhistidine, valine, lysine, and/ormethionine. As a result, one of skill in the art may use NMRspectroscopy to determine whether the quantity of the one or moreacetylated amino acids listed above has increased, e.g., by about 5%,6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 100%, or more, or by about 1.1-fold ormore, such as about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold,1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold,6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold,50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 500-fold,1,000-fold, 10,000-fold, or more, relative to the quantity of thesesubstances in a reference sample, such as a sample isolated from asubject prior to administration of BCG.

As shown in FIG. 5, a subject presenting with a disease described above(e.g., type I diabetes) can additionally be administered BCG in order toregulate methylated metabolite levels. For instance, BCG can beadministered to a subject in order to increase the levels of variousmethylated metabolites, such as N-α-acetyl-3-methylhistidine,3-methylglutaconic acid, 3-methylglutarylcarnitine, andN-ε-trimethyllysine (e.g., by promoting the methylation ofN-α-acetylhistidine, glutaconic acid, glutarylcarnitine, lysine, andcysteine), e.g., by about 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%,35%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, ormore, or by about 1.1-fold or more, such as about 1.1-fold, 1.2-fold,1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold,2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold,20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold,100-fold, 500-fold, 1,000-fold, 10,000-fold, or more, relative to thequantity of these substances in a reference sample, such as a sampleisolated from a subject prior to administration of BCG. Additionally,BCG administration reduces the levels of certain methylated metabolites,such as 4-methyl-2-oxopentanoic acid and 3-methyl-2-oxobutyric acid(e.g., by promoting demethylation of these metabolites) e.g., by about5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more, or by about 1.1-foldor more, such as about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold,1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold,6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold,50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 500-fold,1,000-fold, 10,000-fold, or more, relative to the quantity of thesesubstances in a reference sample, such as a sample isolated from asubject prior to administration of BCG. The levels of these metabolitescan be quantified using techniques known in the art, such as NMRspectroscopy, HPLC, MS, and other standard procedures.

The effect of BCG on protein expression is also manifested in theup-regulation and down-regulation of various enzymes and receptorsinvolved in glucose and lipid metabolism upon administration of BCG to asubject. As the data in FIG. 6 shows, subjects suffering from type Idiabetes that were administered BCG exhibited an increase in variouscytokines (e.g., IL-6, TNFα, and IFNγ) as well as lipolytic factors(e.g., acyl co-enzyme A oxidase, carnitine palmitoyltransferase, lipase,and uncoupling protein). BCG also resulted in a marked decrease invarious adiponectin receptors (e.g., adiponectin receptors 1 and 2) andlipogenic proteins (e.g., uncoupling protein, carnitine palmitoyltransferase A, B, and C, and acetyl-CoA carboxylase 1, 2, and 3). Thus,the levels of these biomarkers can be assessed to determine (1) whethera patient is responsive to BCG treatment, and (2) whether administrationof one or more subsequent doses of BCG is necessary to achieve atherapeutic effect (e.g., to restore the serum concentration ofcholesterol, LDLs, HDLs, triglycerides, and/or HbA1c to a healthy levelin a patient).

Example 3. Diagnosing a Patient Presenting with or Prone to ElevatedCholesterol as Likely to Respond to BCG Therapy

A physician of skill in the art can determine whether a patient (e.g., apatient that has already been diagnosed as having a particular disease,such as elevated cholesterol, LDLs, or triglycerides, reduced HDLlevels, a disease associated with these altered serum lipid levels, oran immunological, neurological, or metabolic disease described herein)is likely to respond to BCG therapy by determining the quantity ofmethylated cytosine residues in a sample isolated from the subject andcomparing this quantity to the amount of methylated cytosine residues inthe DNA sequence of the same genetic locus in a reference sample. Thereference sample may be a sample isolated from a healthy patient,optionally of the same age, sex, and/or weight, or the reference samplemay be a standard quantity of methylated cytosine residues in aparticular DNA sequence that is generally associated with a healthyphysiological state or observed in healthy subjects, such as between 1and 100 methylated cytosine residues (e.g., between 1 and 50 methylatedcytosine residues, between 1 and 25 methylated cytosine residues, orbetween 1 and 10 methylated cytosine residues). A determination that thequantity of methylated cytosine residues in the sample isolated from thepatient is greater than or less than the amount of methylated cytosineresidues in the same DNA sequence within a reference sample (e.g., byabout 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more, or by about1.1-fold or more (e.g., about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold,1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold,4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold,30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold,500-fold, 1,000-fold, 10,000-fold, or more) indicates that the subjectis likely to respond to treatment with a therapeutic agent, such as BCG,in order to treat the disease. In preferred embodiments, the gene thatis analyzed encodes a transcription factor, such as FoxP3, or acell-surface protein, such as CD45. In these cases, a determination thatthe quantity of methylated cytosine residues in the sample isolated fromthe subject is greater than the quantity of methylated cytosine residuesin the same DNA sequence within a reference sample (e.g., by about 5%,6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 100%, or more, or by about 1.1-fold ormore (e.g., about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold,1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold,6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold,50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 500-fold,1,000-fold, 10,000-fold, or more) indicates that the subject is likelyto respond to administration of a therapeutic agent, such as BCG, inorder to treat the disease.

Example 4. Determining the Likelihood that a Patient Presenting withElevated Cholesterol would Benefit from Additional Doses of BCG

A subject that is administered BCG or another therapeutic agent in orderto reduce serum cholesterol levels may be examined using one or moreanalytical tests in order to assess the responsiveness of the subject toadministration of BCG or another therapeutic agent and to determine ifthe subject would benefit from additional doses of BCG or anothermedicament. The subject may be administered BCG or another therapeuticagent and may subsequently have a blood sample withdrawn in order todetermine if the methylation state of one or more cytosine residues inthe nuclear DNA within a cell of the subject has changed in response toadministration of BCG or another therapeutic agent. For instance, aphysician of skill in the art can withdraw a blood sample from a subjecthaving previously been administered BCG or another therapeutic agent andmay analyze the nuclear DNA within a cell of the sample (e.g., within aT-lymphocyte of the sample, such as a CD4+, CD25+T-reg cell) todetermine if one or more cytosine residues has been methylated ordemethylated in response to treatment with BCG or another therapeuticagent. Preferably, the one or more cytosine residues are located withina gene that encodes a transcription factor, such as FoxP3, or within agene that encodes CD45. A determination that the quantity of methylatedcytosine residues in one or more of both of these genetic loci hasdecreased indicates that the patient is responding to treatment with BCGor another therapeutic agent and may not require subsequently doses. Forinstance, a determination that one or more particular cytosine residueswithin the FoxP3 and/or CD45 genes that was previously methylated in thesubject prior to treatment with BCG or another therapeutic agenttreatment and has since undergone demethylation indicates that thesubject is responding to the therapy and may not require subsequentdoses of BCG or another medicament.

In contrast, a determination that the quantity of methylated cytosineresidues in one or both of these genetic loci has stayed the same orincreased indicates that the subject would benefit from one or moreadditional doses of BCG or another therapeutic agent. For example, adetermination that one or more particular cytosine residues within theFoxP3 and/or CD45 genes that was previously methylated in the subjectprior to treatment with BCG or another therapeutic agent and remainsmethylated following administration of BCG or another therapeutic agentindicates that the subject would benefit from one or more subsequentdoses of BCG or another therapeutic agent. Alternatively, adetermination that that one or more particular cytosine residues withinthe FoxP3 and/or CD45 genes that was previously demethylated in thesubject prior to administration of BCG or another therapeutic agent andhas since undergone methylation following administration of BCG oranother therapeutic agent indicates that the subject would benefit fromone or more subsequent doses of BCG or another therapeutic agent.

Example 5. Administering BCG to a Subject in Order to Treat anImmunological Disorder

A subject presenting with an immunological disorder, such as an allergy,can be treated by administering BCG to the subject in a unit dosage formas described herein. For instance, a subject with an allergy, such as afood allergy, seasonal allergy, chemical allergy, or other allergydescribed herein can be treated by administering BCG to the subject in aunit dosage form containing, e.g., between about 1.8×10⁶ and about3.9×10⁶ cfu per 0.1 milligrams of BCG. The subject can subsequently bemonitored by a physician of skill in the art in order to assess theefficacy of BCG in treating the allergy and, if necessary, thelikelihood that the subject would benefit from one or more additionaldoses of BCG. For instance, a physician of skill in the art can withdrawa blood sample from the subject after administering the initial dose ofBCG (e.g., about 1 week, 1 month, 1 year, 5 years, or 10 years after theinitial administration) and can determine the quantity of methylatedcytosine residues in a gene of interest within a cell from the bloodsample, such as a gene encoding FoxP3 or CD45. This quantity can becompared to the quantity of methylated cytosine residues in the samegene of interest within the same cell type of a blood sample isolatedfrom the subject. The cell from which the gene of interest is analyzedcan be, e.g., a T-cell, such as a CD4+, CD25+T-reg cell. A determinationthat the quantity of methylated cytosine residues in the FoxP3 or CD45gene within the sample isolated from the subject after having receivedBCG treatment has decreased (e.g., by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,20, 30, 40, 50, 100, or more residues) relative to the quantity ofmethylated cytosine residues in a sample isolated from the subjectbefore receiving BCG treatment indicates that the subject is respondingwell to the BCG therapy and additional doses may not be needed.Alternatively, a determination that the quantity of methylated cytosineresidues in the gene of interest has stayed the same or has increased(e.g., e.g., by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 100,or more residues) indicates that the subject would likely benefit fromone or more additional doses of BCG.

Additionally or alternatively, the efficacy of BCG in treating theallergy can be assessed by analyzing the level of one or more mRNAmolecules, proteins, acetylated amino acids, and/or methylatedmetabolites in the subject before and after being administered BCG. Forinstance, a physician may withdraw a blood sample from the subject,e.g., about 1 week, 1 month, 1 year, 5 years, or 10 years after theinitial administration of BCG and may determine the level of one or moremRNA molecules encoding a cytokine (such as IL-6, TNFα, or IFNγ) orlipolytic protein (such as acyl co-enzyme A oxidase, carnitinepalmitoyltransferase, lipase, or uncoupling protein). Alternatively, aphysician may directly measure the level of one or more of theseproteins. The level(s) of mRNA molecules and/or proteins recorded forthe sample isolated from the subject after having received BCG can thenbe compared to the level of the same mRNA molecule or protein in asample that was isolated from the subject prior to the subject receivingBCG treatment. The sample that was isolated from the subject prior toreceiving BCG may have been previously analyzed (e.g., a physician mayhave determined the level of the one or more mRNA molecules or proteinsof interest immediately after isolating the sample from the subject), orthe sample may have been preserved (e.g., cryopreserved) and analyzed atthe same time as the sample that was withdrawn following BCG treatment.In either case, a determination that the level(s) of the one or moremRNA molecules and/or proteins has remained the same or has increased(e.g., by about 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more, or byabout 1.1-fold or more (e.g., about 1.1-fold, 1.2-fold, 1.3-fold,1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold,3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold,20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold,100-fold, 500-fold, 1,000-fold, 10,000-fold, or more) following BCGtreatment indicates that the patient would likely benefit from one ormore additional doses of BCG.

If the determination is made that the patient would benefit from one ormore additional dosages of BCG in order to treat the allergy, aphysician of skill in the art can administer BCG to the patient, e.g.,between 1 and 20 additional times, or more, as needed. The physician mayadminister BCG to the patient using a route of administration describedherein, such as intradermally or intravenously. The physician mayadminister BCG to the patient, e.g., between about 1 and about 10 yearsfollowing the initial BCG treatment.

Example 6. Diagnosing a Subject as Having a Disease by Assessing theLevel of a Biomarker and Treating the Subject by Administering BCG

A subject can be diagnosed with a disease, such as an autoimmune diseaseor neurological condition, such as type I diabetes, multiple sclerosis,premature ovarian failure, scleroderma, Sjögren's disease, vitiligo,alopecia, polyglandular failure, Grave's disease, hypothyroidism,polymyositis, pemphigus, Crohn's disease, colitis, autoimmune hepatitis,hypopituitarism, myocarditis, Addison's disease, an autoimmune skindisease, uveitis, pernicious anemia, hypoparathyroidism, and rheumatoidarthritis, using methods described herein. For instance, a subject canbe assessed for the quantity of methylated cytosine residues in anuclear gene, such as FoxP3 or CD45, or for the level of one or moremRNA molecules, proteins, acetylated amino acids, or metabolitesdescribed herein by a physician of skill in the art. If the physiciandetermines that the subject has a particular disease, such as anautoimmune disease (e.g., type I diabetes), the subject can be treatedby administration of BCG. For instance, a physician of skill in the artmay diagnose a subject as having type I diabetes. To render thediagnosis, a physician of skill in the art may withdraw a blood samplefrom the subject and subsequently determine the quantity of methylatedcytosine residues in a gene of interest within a cell from the bloodsample, such as a gene encoding FoxP3 or CD45. This can be accomplishedusing methods known in the art, such as bisulfite-mediated DNAsequencing techniques described herein. This quantity may then have beencompared to the quantity of methylated cytosine residues in the samegene of interest within the same cell type of a blood sample isolatedfrom a healthy subject that does not have type I diabetes (e.g., asubject of the same age, sex, and/or weight). The cell from which thegene of interest is analyzed may be, e.g., a T-cell, such as a CD4+,CD25+T-reg cell. A determination that the quantity of methylatedcytosine residues in the FoxP3 or CD45 gene within the sample isolatedfrom the subject that has type I diabetes is greater than the quantityof methylated cytosine residues in the sample isolated from the healthysubject (e.g., by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50,100, or more residues) may indicate that the subject has the disease,such as type I diabetes. Optionally, this assessment can be confirmedthrough a variety of other procedures known in the art, such as byassessing the blood glucose level of the subject using standard glucosedetection assays. The subject can subsequently be treated by a physicianthat administers BCG to the subject in a unit dosage form containing,e.g., between about 1.8×10⁶ and about 3.9×10⁶ cfu per 0.1 milligrams ofBCG. The subject could then be assessed for potential re-dosingregimens, e.g., as described in Example 5.

Example 7. Case Study of the Effect of BCG on Human Serum Lipid Levels

In order to assess the therapeutic effect of BCG in human patientspresenting with elevated serum lipid levels, a case study was conductedin which patients were administered BCG and the level of serumcholesterol in each patient was subsequently monitored at specific timeintervals.

A first subject (hereinafter “Subject 1”) presented with a serumcholesterol level of about 200 mg/dL prior to being administered BCG.The subject was subsequently administered BCG and the subject's level ofserum cholesterol was assessed five years following this treatment. Atthis point, the subject exhibited a serum cholesterol level of about 150mg/dL. In parallel, a control subject that was not administered BCG wasevaluated at the same time points that were used for assessment ofSubject 1. The control subject exhibited an initial cholesterol level ofabout 150 mg/dL. After five years, the control subject exhibited acholesterol level of about 175 mg/dL.

Similarly, a second subject (“Subject 2”) was initially observed ashaving a serum cholesterol level of about 175 mg/dL. Five yearsfollowing administration of BCG to the subject, the subject exhibited acholesterol level of about 150 mg/dL. A control subject assessed inparallel with Subject 2 exhibited an increase of from about 175 mg/dL toabout 220 mg/dL over the same time period.

The third subject in this study (“Subject 3”) was initially observed ashaving a serum cholesterol level of about 150 mg/dL. Five yearsfollowing administration of BCG to the subject, the subject exhibited acholesterol level of about 125 mg/dL. A control subject assessed inparallel with Subject 3 exhibited an increase of from about 175 mg/dL toabout 220 mg/dL over the same time period.

Taken together, these observations demonstrate that BCG effectivelylowers the concentration of serum cholesterol in patients presentingwith elevated lipid levels, such as patients suffering from type Idiabetes. Each of the subjects and controls described in this case studywere previously diagnosed as having type I diabetes. However, theeffects of BCG on serum lipid levels are not limited to patients withthis disease, as BCG can be used to modulate cholesterol, LDL, HDL,and/or triglyceride levels in patients having elevated serum lipids.

Example 8. Administering BCG to a Subject Prone to the Onset ofHypercholesteremia in Order to Prevent the Development of Elevated SerumCholesterol Levels

BCG can be administered to a subject that is prone to develop elevatedlevels of serum cholesterol even though the patient may not currentlyexhibit elevated serum concentrations of these lipids. For instance, asubject that currently has a serum cholesterol level that is less than129 mg/dL (e.g., about 100 mg/dL, 105 mg/dL, 110 mg/dL, 115 mg/dL, 120mg/dL, or 125 mg/dL, or lower) and that currently has a serumcholesterol level that is within 25% of a previous measurement of theserum cholesterol level of the subject (e.g., a measurement that wasrecorded within the last 1 day to 20 years, such as a measurement thatwas recorded within the last 1-5 years) can be administered BCG by aphysician of skill in the art in order to prevent the subject fromdeveloping an elevated serum cholesterol level in the future. Thesubject may be one that is prone to develop high cholesterol, e.g., asinferred by a prevalence of cases of elevated cholesterol in thesubject's family. The administration of BCG may prevent the subject fromdeveloping an elevated serum cholesterol level, such that upon futureexamination of the subject, e.g., between about 1 day and 20 years, ormore, following the administration (such as about 1 day, 1 week, 1month, 6 months, 1 year, 2 years, 3 years, 4 years, 5 years, 10 years,15 years, or 20 years, or more, following the administration of BCG),the subject may present serum cholesterol levels that are within 25%(e.g., within 10% or less) of the level of serum cholesterol exhibitedby the subject at the time of BCG administration. The subject mayadditionally be assessed for elevated serum cholesterol or diseasesassociated therewith, such as hypercholesterolemia, hyperlipidemia,coronary heart disease, peripheral arterial disease (PAD), peripheralvascular disease, hypertension, stroke, diabetes, metabolic syndrome,obesity, and insulin resistance, before and/or after BCG therapy, byassessing the level of one or more biomarkers in a sample isolated fromthe subject. The biomarkers that are assessed may be one or moremethylated cytosine residues in a nuclear gene of interest, such as agene encoding CD45 or FoxP3. Additionally or alternatively, a physicianof skill in the art may assess the subject's level of cholesterol orneed for additional doses of BCG by analyzing the level of one or morecytokines (such as IL-6, TNFα, or IFNγ) or lipolytic proteins (such asacyl co-enzyme A oxidase, carnitine palmitoyltransferase, lipase, oruncoupling protein). Alternatively, a physician may directly measure thelevel of one or more of these proteins. A determination that the levelsof these cytokines and/or lipolytic proteins, or the levels of mRNAtranscripts encoding these proteins, are within 10% of the levels ofthese biomarkers previously measured in a sample isolated from thesubject (e.g., within 5% or the same as those previously measured in asample isolated from the subject) indicates that the subject hassustained the level of serum cholesterol exhibited prior toadministration of BCG.

Example 9. Administration of BCG Induces a Shift from OxidativePhosphorylation to Aerobic Glycolysis

In order to assess the molecular effects of BCG in human patients and incultured human blood cells, a series of experiments were conducted aimedat understanding how BCG modulates the metabolism of glucose andcholesterol. As shown in FIG. 7, human type-1 diabetes patients treatedwith BCG exhibited a sustained decrease in total cholesterol level overthe course of an 8-year investigation. BCG administration was furthershown to enhance NR1H3 expression at the mRNA level in vitro in culturedperipheral blood lymphocytes and in vivo in human type-1 diabetespatients (FIG. 7). Dissecting the effects of BCG administration furtherrevealed that BCG promotes the expression of cholesterol-suppressinggenes and attenuates the expression of glucose-elevating genes,providing a two-fold therapeutic effect of reducing total cholesteroland blood sugar level. The ability of BCG to modulate blood sugar isalso manifest in the stabilizing effect on glycated hemoglobin levelsobserved upon administration of BCG to human type-1 diabetes patients(FIG. 8).

Surprisingly, I have discovered that BCG is capable of inducing ametabolic conversion from a state of oxidative phosphorylation to astate of aerobic glycolysis (FIG. 9). This conversion results in therapid consumption of glucose. As shown in FIG. 9, the effect of BCGadministration to type-1 diabetic patients on blood glucose is due to anincreased flux through glycolysis and is independent of pancreasregeneration. The ability of BCG to induce a conversion from a state ofoxidative phosphorylation to one of aerobic glycolysis is due to BCG'scapacity to promote HIF1-α expression (FIG. 9).

In further evidence of the ability of BCG to induce a shift towardsglycolysis, I have observed that BCG is capable of up-regulating glucosetransporters and early glycolytic enzymes while suppressing theexpression of proteins involved in the Krebs cycle (FIG. 10).

Example 10. BCG Therapy can Treat Hyperglycemia in any Disease State,Regardless of the Underlying Etiology

The physiological manifestations of the biochemical phenomena describedin Example 9 were further investigated in hyperglycemic BALB/c mice. Astate of hyperglycemia was induced in BALB/c mice by administration ofstreptozotocin, an agent that non-specifically elevates blood glucose.Hyperglycemic mice treated with BCG not only exhibited an improvedability to maintain weight, but also markedly reduced and stabilizedblood sugar levels (FIG. 11). These effects are significant, asstreptozotocin induces an increase in blood glucose in adisease-independent fashion. That BCG therapy was capable of promotingmaintenance of weight and reduction in blood sugar levels instreptozotocin-treated mice indicates that BCG can reduce blood sugar inany disease state, regardless of the underlying etiology.

Other Embodiments

All publications, patents, and patent applications mentioned in thisspecification are incorporated herein by reference to the same extent asif each independent publication or patent application was specificallyand individually indicated to be incorporated by reference.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from theinvention that come within known or customary practice within the art towhich the invention pertains and may be applied to the essentialfeatures hereinbefore set forth, and follows in the scope of the claims.

Other embodiments are within the claims.

1. A method of reducing the level of cholesterol, low-densitylipoprotein (LDL), or triglycerides in a subject in need thereofcomprising administering Bacillus Calmette-Guerin (BCG) to said subject.2. A method of increasing the level of high-density lipoprotein (HDL) ina subject in need thereof comprising administering BCG to said subject.3. The method of claim 1 or 2, whereby said administering lowers thelevel of total cholesterol in said subject by 5% or more.
 4. The methodof claim 3, wherein said subject has a total cholesterol level of about100 mg/dL or greater, preferably wherein said subject has a totalcholesterol level of about 129 mg/dL or greater.
 5. The method of anyone of claims 1-4, whereby said administering lowers the level of LDLsin said subject by 5% or more.
 6. The method of claim 5, wherein saidsubject has a LDL level of about 80 mg/dL or greater.
 7. The method ofany one of claims 1-6, whereby said administering elevates the level ofhigh-density lipoproteins (HDLs) in said subject by 5% or more.
 8. Themethod of claim 7, wherein said subject has a HDL level of about 40mg/dL or below.
 9. The method of any one of claims 1-8, wherein saidsubject has a triglyceride level of about 100 mg/dL or greater.
 10. Themethod of claim 9, wherein said subject has a triglyceride level ofbetween about 100 mg/dL and about 500 mg/dL.
 11. The method of any oneof claims 1-10, wherein said subject has a ratio of total cholesterollevel to HDL level of about 5 or greater.
 12. The method of any one ofclaims 1-10, wherein said subject has a ratio of total cholesterol levelto HDL level of between about 3 and about
 10. 13. The method of any oneof claims 1-3, wherein said subject has a total cholesterol, LDL and/ortriglyceride level that is higher than that which has previously beenobserved for said subject.
 14. The method of claim 13, wherein saidsubject has previously been observed as having a total cholesterol levelless than about 180 mg/dL.
 15. The method of claim 13 or 14, whereinsaid subject has previously been observed as having a LDL level of lessthan about 100 mg/dL.
 16. The method of any one of claims 13-15, whereinsaid subject has previously been observed as having a triglyceride levelof less than about 150 mg/dL.
 17. The method of any one of claims 1-16,wherein said subject is suffering from a disease associated with anelevated level of cholesterol.
 18. The method of claim 17, wherein saiddisease is selected from the group consisting of hypercholesterolemia,hyperlipidemia, coronary heart disease, peripheral arterial disease(PAD), peripheral vascular disease, hypertension, stroke, diabetes,metabolic syndrome, obesity, and insulin resistance.
 19. The method ofclaim 17 or 18, wherein said BCG is administered to said subject in anamount sufficient to alleviate or reduce a symptom associated with saiddisease.
 20. The method of claim 19, wherein said symptom is an elevatedlevel of a substance selected from the group consisting of lactatedehydrogenase (LDH), LDL, and triglycerides.
 21. The method of claim 20,wherein said method results in a reduction in the level of saidsubstance.
 22. The method of claim 19, wherein said symptom is selectedfrom the group consisting of angina, arrhythmia, and heart failure. 23.The method of claim 22, wherein said method alleviates or reduces saidangina, arrhythmia, or heart failure.
 24. The method of any one ofclaims 1-23, wherein said method further comprises administering to saidsubject a hypolipidemic agent.
 25. The method of claim 24, wherein saidhypolipidemic agent is selected from the group consisting of a HMG-CoAreductase inhibitor, niacin, a fibric acid derivative, a cholesterolabsorption inhibitor, and a lipolytic agent.
 26. The method of claim 25,wherein said HMG-CoA reductase inhibitor is selected from the groupconsisting of atorvastatin, cerivastatin, fluvastatin, lovastatin,mevastatin, pitavastatin, pravastatin, rosuvastatin, simvastatin, andcombinations thereof.
 27. The method of claim 25, wherein said fibricacid derivative is selected from the group consisting of fenofibrate andgemfibrozil.
 28. The method of claim 25, wherein said cholesterolabsorption inhibitor is ezetimibe.
 29. The method of claim 25, whereinsaid lipolytic agent is selected from the group consisting ofnorepinephrine, isoproterenol, forskolin, bucladesine, and theophylline.30. A method of treating a subject that has previously been diagnosed ashaving a disease selected from the group consisting of an autoimmunedisease and a neurological condition, wherein said subject has beenpreviously diagnosed by: a) determining a quantity of methylatedcytosine residues in a sample of nuclear DNA isolated from said subject;and b) comparing said quantity to a quantity of methylated cytosineresidues in a reference sample, wherein a determination that thequantity of methylated cytosine residues in said sample of nuclear DNAisolated from said subject is greater than or less than the quantity ofmethylated cytosine residues in said reference sample indicates that thesubject has said disease, said method comprising administering BCG tosaid subject.
 31. A method of treating a subject that has previouslybeen diagnosed as having a disease selected from the group consisting ofan autoimmune disease and a neurological condition, wherein said subjecthas been previously diagnosed by: a) i) determining a level of one ormore substances selected from the group consisting of an mRNA moleculeencoding a cytokine or lipolytic protein, a protein selected from thegroup consisting of a cytokine and a lipolytic protein, an acetylatedamino acid selected from the group consisting of N-acetylalanine,N-acetylaspartic acid, N-acetylserine, N-acetylthreonine,N-acetylhistidine, N-acetyl-3-methylhistidine, N-acetylvaline, andN-α-acetyllysine, and N-acetylmethionine, or a methylated metaboliteselected from the group consisting of N-α-acetyl-3-methylhistidine,3-methylglutaconic acid, 3-methylglutarylcarnitine, andN-ε-trimethyllysine in a sample from said subject; and (ii) comparingthe level of the one or more substances to the level of the one or moresubstances in a reference sample, wherein a determination that the levelof said one or more substances in the sample from the subject havingsaid disease is less than the level of said one or more substances inthe reference sample indicates that the subject has said disease; or b)(i) determining a level of one or more substances selected from thegroup consisting of an mRNA molecule encoding a lipogenic protein, anadiponectin receptor, a lysine acetyltransferase, a histoneacetyltransferase, a histone deacetylase, or a histone, a proteinselected form the group consisting of a lipogenic protein, anadiponectin receptor, a lysine acetyltransferase, a histoneacetyltransferase, a histone deacetylase, and a histone, or a methylatedmetabolite selected from the group consisting of 4-methyl-2-oxopentanoicacid and 3-methyl-2-oxobutyric acid in a sample from said subject; and(ii) comparing the level of the one or more substances to the level ofthe one or more substances in a reference sample, wherein adetermination that the level of said one or more substances in thesample from the subject having said disease is greater than the level ofsaid one or more substances in the reference sample indicates that thesubject has said disease, said method comprising administering BCG tosaid subject.
 32. The method of claim 30 or 31, wherein said autoimmunedisease is selected from the group consisting of type I diabetes,Alopecia Areata, Ankylosing Spondylitis, Antiphospholipid Syndrome,Autoimmune Addison's Disease, Autoimmune Hemolytic Anemia, AutoimmuneHepatitis, Behcet's Disease, Bullous Pemphigoid, Cardiomyopathy, CeliacSprue-Dermatitis, Chronic Fatigue Immune Dysfunction Syndrome (CFIDS),Chronic Inflammatory Demyelinating Polyneuropathy, Churg-StraussSyndrome, Cicatricial Pemphigoid, CREST Syndrome, Cold AgglutininDisease, Crohn's Disease, Essential Mixed Cryoglobulinemia,Fibromyalgia-Fibromyositis, Graves' Disease, Guillain-Barré, Hashimoto'sThyroiditis, Hypothyroidism, Idiopathic Pulmonary Fibrosis, IdiopathicThrombocytopenia Purpura (ITP), IgA Nephropathy, Juvenile Arthritis,Lichen Planus, Lupus, Ménière's Disease, Mixed Connective TissueDisease, Multiple Sclerosis, Myasthenia Gravis, Pemphigus Vulgaris,Pernicious Anemia, Polyarteritis Nodosa, Polychondritis, PolyglandularSyndromes, Polymyalgia Rheumatica, Polymyositis and Dermatomyositis,Primary Agammaglobulinemia, Primary Biliary Cirrhosis, Psoriasis,Raynaud's Phenomenon, Reiter's Syndrome, Rheumatic Fever, RheumatoidArthritis, Sarcoidosis, Scleroderma, Sjögren's Syndrome, Stiff-ManSyndrome, Takayasu Arteritis, Temporal Arteritis/Giant Cell Arteritis,Ulcerative Colitis, Uveitis, Vasculitis, Vitiligo, and Wegener'sGranulomatosis.
 33. The method of claim 30 or 31, wherein saidneurological condition is selected from the group consisting of a braintumor, a brain metastasis, a spinal cord injury, schizophrenia,epilepsy, Amyotrophic lateral sclerosis (ALS), Parkinson's disease,Autism, Alzheimer's disease, Huntington's disease, and stroke.
 34. Amethod of treating a subject having a disease selected from the groupconsisting of a brain tumor, a brain metastasis, schizophrenia,epilepsy, Autism, stroke, an allergy, allograft rejection,graft-versus-host disease, asthma, macular degeneration, muscularatrophy, a disease related to miscarriage, atherosclerosis, bone loss, amusculoskeletal disease, and obesity comprising administering BCG tosaid subject.
 35. The method of claim 34, wherein said allergy isselected from the group consisting of food allergy, seasonal allergy,pet allergy, hives, hay fever, allergic conjunctivitis, poison ivyallergy oak allergy, mold allergy, drug allergy, dust allergy, cosmeticallergy, and chemical allergy.
 36. The method of claim 34, wherein saidallograft rejection is selected from the group consisting of skin graftrejection, bone graft rejection, vascular tissue graft rejection,ligament graft rejection, and organ graft rejection.
 37. The method ofclaim 36, wherein said ligament graft rejection is selected from thegroup consisting of cricothyroid ligament graft rejection, periodontalligament graft rejection, suspensory ligament of the lens graftrejection, palmar radiocarpal ligament graft rejection, dorsalradiocarpal ligament graft rejection, ulnar collateral ligament graftrejection, radial collateral ligament graft rejection, suspensoryligament of the breast graft rejection, anterior sacroiliac ligamentgraft rejection, posterior sacroiliac ligament graft rejection,sacrotuberous ligament graft rejection, sacrospinous ligament graftrejection, inferior pubic ligament graft rejection, superior pubicligament graft rejection, anterior cruciate ligament graft rejection,lateral collateral ligament graft rejection, posterior cruciate ligamentgraft rejection, medial collateral ligament graft rejection, cranialcruciate ligament graft rejection, caudal cruciate ligament graftrejection, and patellar ligament graft rejection.
 38. The method ofclaim 36, wherein said organ graft rejection is selected from the groupconsisting of heart graft rejection, lung graft rejection, kidney graftrejection, liver graft rejection, pancreas graft rejection, intestinegraft rejection, and thymus graft rejection.
 39. The method of claim 34,wherein said graft-versus-host disease arises from a bone marrowtransplant or transplant of one or more blood cells selected from thegroup consisting of hematopoietic stem cells, common myeloid progenitorcells, common lymphoid progenitor cells, megakaryocytes, monocytes,basophils, eosinophils, neutrophils, macrophages, T-cells, B-cells,natural killer cells, and dendritic cells.
 40. The method of any one ofclaims 1-39, wherein said method further comprises administering to saidsubject an additional therapeutic agent.
 41. The method of claim 40,wherein said additional therapeutic agent is selected from the groupconsisting of tumor necrosis factor-alpha (TNFα), a tumor necrosisfactor receptor 2 (TNFR2) agonist, an immunotherapy agent, andcombinations thereof.
 42. The method of claim 41, wherein saidimmunotherapy agent is selected from the group consisting of ananti-CTLA-4 agent, an anti-PD-1 agent, an anti-PD-L1 agent, ananti-PD-L2 agent, a TNFα cross-linking agent, a TRAIL cross-linkingagent, a CD27 agent, a CD30 agent, a CD40 agent, a 4-1 BB agent, a GITRagent, an OX40 agent, a TRAILR1 agent, a TRAILR2 agent, and a TWEAKRagent.
 43. The method of any one of claims 1-42, wherein said BCG isadministered in a unit dosage form comprising between about 5×10⁵ andabout 1×10⁷ colony forming units (cfu) per 0.1 milligrams of BCG. 44.The method of claim 43, wherein said unit dosage form comprises betweenabout 1×10⁶ and about 6×10⁶ cfu per 0.1 milligrams of BCG.
 45. Themethod of claim 44, wherein said unit dosage form comprises betweenabout 1.8×10⁶ and about 3.9×10⁶ cfu per 0.1 milligrams of BCG.
 46. Themethod of any one of claims 1-45, whereby said administering modulates amethylation state of one or more deoxyribonucleotides in said subject.47. The method of claim 46, whereby said administering promotesmethylation of one or more deoxyribonucleotides in said subject.
 48. Themethod of claim 46, whereby said administering promotes demethylation ofone or more deoxyribonucleotides in said subject.
 49. The method ofclaim 48, wherein said one or more deoxyribonucleotides are locatedwithin a gene that encodes a transcription factor.
 50. The method ofclaim 49, wherein said transcription factor is FoxP3.
 51. The method ofclaim 48, wherein said one or more deoxyribonucleotides are locatedwithin a gene that encodes a protein that is expressed on the surface ofa T-cell.
 52. The method of claim 51, wherein said protein is CD45. 53.The method of any one of claims 46-52, wherein said one or moredeoxyribonucleotides comprise cytosine.
 54. The method of any one ofclaims 1-53, whereby said administering promotes an increase in thelevel of one or more mRNA molecules that encode one or more proteins insaid subject.
 55. The method of any one of claims 1-53, whereby saidadministering promotes an increase in the level of one or more proteinsin said subject.
 56. The method of claim 54 or 55, wherein said one ormore proteins comprise a cytokine.
 57. The method of claim 56, whereinsaid cytokine is selected from the group consisting of interleukin-6(IL-6), tumor necrosis factor (TNFα), and interferon-gamma (IFNγ). 58.The method of claim 54 or 55, wherein said one or more proteins comprisea lipolytic protein.
 59. The method of claim 58, wherein said lipolyticprotein is selected from the group consisting of acyl co-enzyme Aoxidase, carnitine palmitoyltransferase, lipase, and uncoupling protein.60. The method of any one of claims 1-53, whereby said administeringpromotes a decrease in the level of one or more mRNA molecules thatencode one or more proteins in said subject.
 61. The method of any oneof claims 1-53, whereby said administering promotes a decrease in thelevel of one or more proteins in said subject.
 62. The method of claim60 or 61, wherein said one or more proteins comprise a lipogenicprotein.
 63. The method of claim 62, wherein said lipogenic protein isselected from the group consisting of acetyl co-enzyme A carboxylase α,acetyl co-enzyme A carboxylase β, fatty acid synthase,glyceraldehydes-6-phosphate dehydrogenase, stearoyl-CoA saturase, malicenzyme, and glucose-6-phosphate dehydrogenase.
 64. The method of claim60 or 61, wherein said one or more proteins comprise an adiponectinreceptor.
 65. The method of claim 64, wherein said adiponectin receptoris selected from the group consisting of adiponectin receptor 1 andadiponectin receptor
 2. 66. The method of any one of claims 1-65,whereby said administering promotes acetylation of one or more aminoacids in said subject.
 67. The method of claim 66, wherein said aminoacid is selected from the group consisting of alanine, aspartic acid,serine, threonine, histidine, 3-methylhistidine, valine, lysine, andmethionine.
 68. The method of any one of claims 1-67, whereby saidadministering promotes methylation of one or more metabolites in saidsubject.
 69. The method of claim 68, wherein said metabolite is selectedfrom the group consisting of N-α-acetylhistidine, glutaconic acid,glutarylcarnitine, lysine, and cysteine.
 70. The method of any one ofclaims 1-69, whereby said administering promotes demethylation of one ormore metabolites in said subject.
 71. The method of claim 70, whereinsaid metabolite is selected from the group consisting of4-methyl-2-oxopentanoic acid and 3-methyl-2-oxobutyric acid.
 72. Themethod of any one of claims 1-71, whereby said administering promotes adecrease in the level of one or more lysine acetyltransferases (KATs).73. The method of claim 72, wherein said one or more lysineacetyltransferases are selected from the group consisting of KAT2A,KAT2B, KAT5, KAT6A, KAT6B, KAT7, and KAT8.
 74. The method of any one ofclaims 1-73, whereby said administering promotes a decrease in the levelof one or more histone acetyltransferases.
 75. The method of any one ofclaims 1-74, whereby said administering promotes a decrease in the levelof one or more histone deacetylases (HDACs).
 76. The method of claim 75,wherein said one or more histone deacetylases are selected form thegroup consisting of HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7,HDAC8, HDAC9, HDAC10, HDAC11, HDAC1P1, HDAC1P2, SIRT1, SIRT2, SIRT3,SIRT4, SIRT5, SIRT6, and SIRT7.
 77. The method of any one of claims1-76, whereby said administering promotes a decrease in the level of oneor more histones.
 78. The method of claim 77, wherein said one or morehistones belong to a family selected from the group consisting of H2A,H2B, H3, and H4.
 79. The method of any one of claims 1-78, wherein saidsubject exhibits a reduction in total cholesterol of between about 5%and about 40% relative to a control subject not treated with said BCG.80. The method of claim 79, wherein said subject exhibits said reductionin total cholesterol within about 3 months to about 7 years after beingtreated with said BCG.
 81. The method of any one of claims 1-80, whereinsaid subject exhibits a reduction in LDL of between about 5% and about60%.
 82. The method of claim 81, wherein said subject exhibits saidreduction in LDL within about 3 months to about 7 years after beingtreated with said BCG.
 83. The method of any one of claims 1-82, whereinsaid subject exhibits a reduction in glycated hemoglobin of betweenabout 5% and about 30%.
 84. The method of claim 83, wherein said subjectexhibits said reduction in glycated hemoglobin within about 2 weeks toabout 7 years after being treated with said BCG.
 85. The method of anyone of claims 79-84, wherein said reduction is maintained for betweenabout 1 year and about 8 years.
 86. A method of diagnosing a subject ashaving a disease, said method comprising: a) determining a quantity ofmethylated cytosine residues in a sample of nuclear DNA isolated fromsaid subject; and b) comparing said quantity to a quantity of methylatedcytosine residues in a reference sample, wherein a determination thatthe quantity of methylated cytosine residues in said sample of nuclearDNA isolated from said subject is greater than or less than the quantityof methylated cytosine residues in said reference sample indicates thatthe subject has said disease.
 87. The method of claim 86, said methodfurther comprising determining whether the subject is likely to respondto treatment with a therapeutic agent for said disease, wherein adetermination that the quantity of methylated cytosine residues in saidsample of nuclear DNA isolated from said subject is greater than or lessthan the quantity of methylated cytosine residues in said referencesample indicates that said subject is likely to respond to saidtreatment.
 88. The method of claim 86 or 67, wherein said quantity ofmethylated cytosine residues in said sample of nuclear DNA isolated fromsaid subject is greater than said quantity of methylated cytosineresidues in said reference sample by 1% or more.
 89. The method of claim88, wherein said quantity of methylated cytosine residues in said sampleof nuclear DNA isolated from said subject is greater than said quantityof methylated cytosine residues in said reference sample by 5% or more.90. The method of claim 89, wherein said quantity of methylated cytosineresidues in said sample of nuclear DNA isolated from said subject isgreater than said quantity of methylated cytosine residues in saidreference sample by 10% or more.
 91. The method of claim 86 or 87,wherein said quantity of methylated cytosine residues in said sample ofnuclear DNA isolated from said subject is less than said quantity ofmethylated cytosine residues in said reference sample by 1% or more. 92.The method of claim 91, wherein said quantity of methylated cytosineresidues in said sample of nuclear DNA isolated from said subject isless than said quantity of methylated cytosine residues in saidreference sample by 5% or more.
 93. The method of claim 92, wherein saidquantity of methylated cytosine residues in said sample of nuclear DNAisolated from said subject is less than said quantity of methylatedcytosine residues in said reference sample by 10% or more.
 94. A methodof determining whether a subject previously administered a therapeuticagent for the treatment of a disease would benefit from receiving one ormore additional doses of said therapeutic agent, said method comprising:a) determining a quantity of methylated cytosine residues in a sample ofnuclear DNA isolated from said subject; and b) comparing said quantityto a quantity of methylated cytosine residues in a reference sample,wherein a determination that the quantity of methylated cytosineresidues in said sample of nuclear DNA isolated from said subject iswithin 10% of the quantity of methylated cytosine residues in saidreference sample indicates that the subject would benefit from one ormore additional doses of said therapeutic agent.
 95. The method of claim94, wherein a determination that the quantity of methylated cytosineresidues in said sample of nuclear DNA isolated from said subject iswithin 5% of the quantity of methylated cytosine residues in saidreference sample indicates that the subject would benefit from one ormore additional doses of said therapeutic agent.
 96. The method of claim95, wherein a determination that the quantity of methylated cytosineresidues in said sample of nuclear DNA isolated from said subject iswithin 1% of the quantity of methylated cytosine residues in saidreference sample indicates that the subject would benefit from one ormore additional doses of said therapeutic agent.
 97. The method of claim96, wherein a determination that the quantity of methylated cytosineresidues in said sample of nuclear DNA isolated from said subject is thesame as the quantity of methylated cytosine residues in said referencesample indicates that the subject would benefit from one or moreadditional doses of said therapeutic agent.
 98. The method of any one ofclaims 86-90 and 94-97, wherein said methylated cytosine residues arelocated within a gene that encodes a transcription factor.
 99. Themethod of claim 98, wherein said transcription factor is FoxP3.
 100. Themethod of any one of claims 86-90 and 94-97, wherein said methylatedcytosine residues are located within a gene that encodes a protein thatis expressed on the surface of a T-cell.
 101. The method of claim 100,wherein said protein is CD45.
 102. The method of any one of claims86-101, said method comprising isolating a polynucleotide comprising oneor more cytosine residues and treating said polynucleotide withbisulfite.
 103. The method of claim 102, said method further comprisingamplifying said polynucleotide using a polymerase chain reaction (PCR).104. A method of diagnosing a subject as having a disease, said methodcomprising: a) i) determining a level of one or more substances selectedfrom the group consisting of an mRNA molecule encoding a cytokine orlipolytic protein, a protein selected from the group consisting of acytokine and a lipolytic protein, an acetylated amino acid selected fromthe group consisting of N-acetylalanine, N-acetylaspartic acid,N-acetylserine, N-acetylthreonine, N-acetylhistidine,N-acetyl-3-methylhistidine, N-acetylvaline, and N-α-acetyllysine, andN-acetylmethionine, or a methylated metabolite selected from the groupconsisting of N-α-acetyl-3-methylhistidine, 3-methylglutaconic acid,3-methylglutarylcarnitine, and N-ε-trimethyllysine in a sample from saidsubject; and (ii) comparing the level of the one or more substances tothe level of the one or more substances in a reference sample, wherein adetermination that the level of said one or more substances in thesample from the subject having said disease is less than the level ofsaid one or more substances in the reference sample indicates that thesubject has said disease; or b) (i) determining a level of one or moresubstances selected from the group consisting of an mRNA moleculeencoding a lipogenic protein, an adiponectin receptor, a lysineacetyltransferase, a histone acetyltransferase, a histone deacetylase,or a histone, a protein selected form the group consisting of alipogenic protein, an adiponectin receptor, a lysine acetyltransferase,a histone acetyltransferase, a histone deacetylase, and a histone, or amethylated metabolite selected from the group consisting of4-methyl-2-oxopentanoic acid and 3-methyl-2-oxobutyric acid in a samplefrom said subject; and (ii) comparing the level of the one or moresubstances to the level of the one or more substances in a referencesample, wherein a determination that the level of said one or moresubstances in the sample from the subject having said disease is greaterthan the level of said one or more substances in the reference sampleindicates that the subject has said disease.
 105. The method of claim104, said method further comprising determining whether the subject islikely to respond to treatment with a therapeutic agent for saiddisease, wherein a determination that the level of the one or moresubstances listed in (a) in the sample from the subject having saiddisease is less than the level of said one or more substances in thereference sample indicates that the subject is likely to respond to saidtreatment, or wherein a determination that the level of the one or moresubstances listed in (b) in the sample from the subject having saiddisease is greater than the level of said one or more substances in thereference sample indicates that the subject is likely to respond to saidtreatment.
 106. A method of determining whether a subject previouslyadministered a therapeutic agent for the treatment of a disease wouldbenefit from receiving one or more additional doses of said therapeuticagent, said method comprising: a) (i) determining a level of one or moresubstances selected from the group consisting of a an mRNA moleculeencoding a cytokine or lipolytic protein, a protein selected from thegroup consisting of a cytokine and a lipolytic protein, an acetylatedamino acid selected from the group consisting of N-acetylalanine,N-acetylaspartic acid, N-acetylserine, N-acetylthreonine,N-acetylhistidine, N-acetyl-3-methylhistidine, N-acetylvaline, andN-α-acetyllysine, and N-acetylmethionine, or a methylated metaboliteselected from the group consisting of N-α-acetyl-3-methylhistidine,3-methylglutaconic acid, 3-methylglutarylcarnitine, andN-ε-trimethyllysine in a sample from said subject; and (ii) comparingthe level of the one or more substances to the level of the one or moresubstances in a reference sample, wherein a determination that the levelof said one or more substances in the sample from the subject is thesame as or less than the level of said one or more substances in saidreference sample indicates that said subject would benefit fromadditional doses of said therapeutic agent, or b) (i) determining alevel of one or more substances selected from the group consisting of anmRNA molecule encoding a lipogenic protein, an adiponectin receptor, alysine acetyltransferase, a histone acetyltransferase, a histonedeacetylase, or a histone, a protein selected form the group consistingof a lipogenic protein, an adiponectin receptor, a lysineacetyltransferase, a histone acetyltransferase, a histone deacetylase,and a histone, or a methylated metabolite selected from the groupconsisting of 4-methyl-2-oxopentanoic acid and 3-methyl-2-oxobutyricacid in a sample from said subject; and (ii) comparing the level of theone or more substances to the level of the one or more substances in areference sample, wherein a determination that the level of said one ormore substances in the sample from the subject is the same as or greaterthan the level of said one or more substances in said reference sampleindicates that said subject would benefit from additional doses of saidtherapeutic agent.
 107. The method of any one of claims 104-06, saidmethod comprising determining the level of an mRNA molecule encoding acytokine or lipolytic protein.
 108. The method of any one of claims104-106, said method comprising determining the level of a proteinselected from the group consisting of a cytokine and a lipolyticprotein.
 109. The method of claim 107 or 108, wherein said cytokine isselected from the group consisting of IL-6, TNFα, and IFNγ.
 110. Themethod of claim 107 or 108, wherein said lipolytic protein is selectedfrom the group consisting of acyl co-enzyme A oxidase, carnitinepalmitoyltransferase, lipase, and uncoupling protein.
 111. The method ofany one of claims 104-106, said method comprising determining the levelof an acetylated amino acid selected from the group consisting ofN-acetylalanine, N-acetylaspartic acid, N-acetylserine,N-acetylthreonine, N-acetylhistidine, N-acetyl-3-methylhistidine,N-acetylvaline, and N-α-acetyllysine, and N-acetylmethionine.
 112. Themethod of any one of claims 104-106, said method comprising determiningthe level of a methylated metabolite selected from the group consistingof N-α-acetyl-3-methylhistidine, 3-methylglutaconic acid,3-methylglutarylcarnitine, and N-ε-trimethyllysine.
 113. The method ofany one of claims 104-106, said method comprising determining the levelof an mRNA molecule encoding a lipogenic protein, an adiponectinreceptor, a lysine acetyltransferase, a histone acetyltransferase, ahistone deacetylase, or a histone.
 114. The method of any one of claims104-106, said method comprising determining the level of a proteinselected from the group consisting of a lipogenic protein, anadiponectin receptor, a lysine acetyltransferase, a histoneacetyltransferase, a histone deacetylase, and a histone.
 115. The methodof claim 113 or 114, wherein said lipogenic protein is selected from thegroup consisting of acetyl co-enzyme A carboxylase α, acetyl co-enzyme Acarboxylase β, fatty acid synthase, glyceraldehydes-6-phosphatedehydrogenase, stearoyl-CoA saturase, malic enzyme, andglucose-6-phosphate dehydrogenase.
 116. The method of claim 113 or 114,wherein said adiponectin receptor is selected from the group consistingof adiponectin receptor 1 and adiponectin receptor
 2. 117. The method ofclaim 113 or 114, wherein said lysine acetyltransferase is selected fromthe group consisting of KAT2A, KAT2B, KAT5, KAT6A, KAT6B, KAT7, andKAT8.
 118. The method of claim 113 or 114, wherein said histonedeacetylase is selected from the group consisting of HDAC1, HDAC2,HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11,HDAC1P1, HDAC1P2, SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, and SIRT7.119. The method of claim 113 or 114, wherein said histone is selectedfrom the group consisting of H2A, H2B, H3, and H4.
 120. The method ofany one of claims 104-106, said method comprising determining the levelof a methylated metabolite selected from the group consisting of4-methyl-2-oxopentanoic acid and 3-methyl-2-oxobutyric acid.
 121. Themethod of any one of claims 104-106, 107, 109, 110, 113, and 115-119,wherein the level of said mRNA molecule is determined by performing anassay selected from the group consisting of reverse transcription PCR(RT-PCR) and a Northern blot.
 122. The method of any one of claims104-106, 108-110, and 114-119, wherein the level of said protein isdetermined by performing an assay selected from the group consisting ofan immunoblot and an enzyme-linked immunosorbant assay (ELISA).
 123. Themethod of any one of claims 104-106, 111, 112, and 120, wherein thelevel of said acetylated amino acid or said methylated metabolite isdetermined by nuclear magnetic resonance (NMR) spectroscopy.
 124. Themethod of any one of claims 30, 31, 86-93, 104, and 105, wherein saidreference sample is a sample isolated from a control subject not havingsaid disease.
 125. The method of claim 124, wherein said control subjectis a subject of the same age and/or gender of said subject having saiddisease.
 126. The method of any one of 94-97 and 106, wherein saidreference sample is a prior sample that has been previously isolatedfrom said subject.
 127. The method of claim 126, wherein said priorsample was isolated between about 24 hours and about 5 years beforemaking said determination.
 128. The method of claim 127, wherein saidprior sample was isolated between about 1 month and about 1 year priorto making said determination.
 129. The method of any one of claims126-128, wherein said prior sample was isolated prior to said subjectbeing administered said therapeutic agent.
 130. The method of any one ofclaims 86-129, wherein said disease is a condition associated withelevated levels of cholesterol.
 131. The method of 130, wherein saidcondition is selected from the group consisting of hypercholesterolemia,hyperlipidemia, coronary heart disease, peripheral arterial disease(PAD), peripheral vascular disease, hypertension, stroke, diabetes,metabolic syndrome, obesity, and insulin resistance.
 132. The method ofany one of claims 86-129, wherein said disease is selected from thegroup consisting of an autoimmune disease, a neurological condition, anallergy, allograft rejection, graft-versus-host disease, asthma, maculardegeneration, muscular atrophy, a disease related to miscarriage,atherosclerosis, bone loss, a musculoskeletal disease, and obesity. 133.The method of claim 132, wherein said disease associated with elevatedlevels of cholesterol is selected from the group consisting ofhypercholesterolemia, coronary heart disease, peripheral vasculardisease, hypertension, and stroke.
 134. The method of claim 132, whereinsaid autoimmune disease is selected from the group consisting of type Idiabetes, Alopecia Areata, Ankylosing Spondylitis, AntiphospholipidSyndrome, Autoimmune Addison's Disease, Autoimmune Hemolytic Anemia,Autoimmune Hepatitis, Behcet's Disease, Bullous Pemphigoid,Cardiomyopathy, Celiac Sprue-Dermatitis, Chronic Fatigue ImmuneDysfunction Syndrome (CFIDS), Chronic Inflammatory DemyelinatingPolyneuropathy, Churg-Strauss Syndrome, Cicatricial Pemphigoid, CRESTSyndrome, Cold Agglutinin Disease, Crohn's Disease, Essential MixedCryoglobulinemia, Fibromyalgia-Fibromyositis, Graves' Disease,Guillain-Barré, Hashimoto's Thyroiditis, Hypothyroidism, IdiopathicPulmonary Fibrosis, Idiopathic Thrombocytopenia Purpura (ITP), IgANephropathy, Juvenile Arthritis, Lichen Planus, Lupus, Ménière'sDisease, Mixed Connective Tissue Disease, Multiple Sclerosis, MyastheniaGravis, Pemphigus Vulgaris, Pernicious Anemia, Polyarteritis Nodosa,Polychondritis, Polyglandular Syndromes, Polymyalgia Rheumatica,Polymyositis and Dermatomyositis, Primary Agammaglobulinemia, PrimaryBiliary Cirrhosis, Psoriasis, Raynaud's Phenomenon, Reiter's Syndrome,Rheumatic Fever, Rheumatoid Arthritis, Sarcoidosis, Scleroderma,Sjögren's Syndrome, Stiff-Man Syndrome, Takayasu Arteritis, TemporalArteritis/Giant Cell Arteritis, Ulcerative Colitis, Uveitis, Vasculitis,Vitiligo, and Wegener's Granulomatosis.
 135. The method of claim 132,wherein said neurological condition is selected from the groupconsisting of a brain tumor, a brain metastasis, a spinal cord injury,schizophrenia, epilepsy, Amyotrophic lateral sclerosis (ALS),Parkinson's disease, Autism, Alzheimer's disease, Huntington's disease,and stroke.
 136. The method of claim 132, wherein said allergy isselected from the group consisting of food allergy, seasonal allergy,pet allergy, hives, hay fever, allergic conjunctivitis, poison ivyallergy oak allergy, mold allergy, drug allergy, dust allergy, cosmeticallergy, and chemical allergy.
 137. The method of claim 132, whereinsaid allograft rejection is selected from the group consisting of skingraft rejection, bone graft rejection, vascular tissue graft rejection,ligament graft rejection, and organ graft rejection.
 138. The method ofclaim 137, wherein said ligament graft rejection is selected from thegroup consisting of cricothyroid ligament graft rejection, periodontalligament graft rejection, suspensory ligament of the lens graftrejection, palmar radiocarpal ligament graft rejection, dorsalradiocarpal ligament graft rejection, ulnar collateral ligament graftrejection, radial collateral ligament graft rejection, suspensoryligament of the breast graft rejection, anterior sacroiliac ligamentgraft rejection, posterior sacroiliac ligament graft rejection,sacrotuberous ligament graft rejection, sacrospinous ligament graftrejection, inferior pubic ligament graft rejection, superior pubicligament graft rejection, anterior cruciate ligament graft rejection,lateral collateral ligament graft rejection, posterior cruciate ligamentgraft rejection, medial collateral ligament graft rejection, cranialcruciate ligament graft rejection, caudal cruciate ligament graftrejection, and patellar ligament graft rejection.
 139. The method ofclaim 137, wherein said organ graft rejection is selected from the groupconsisting of heart graft rejection, lung graft rejection, kidney graftrejection, liver graft rejection, pancreas graft rejection, intestinegraft rejection, and thymus graft rejection.
 140. The method of claim132, wherein said graft-versus-host disease arises from a bone marrowtransplant or transplant of one or more blood cells selected from thegroup consisting of hematopoietic stem cells, common myeloid progenitorcells, common lymphoid progenitor cells, megakaryocytes, monocytes,basophils, eosinophils, neutrophils, macrophages, T-cells, B-cells,natural killer cells, and dendritic cells.
 141. The method of any one ofclaims 87-103 and 104-140, wherein said therapeutic agent is BCG. 142.The method of any one of claims 86-141, said method further comprisingadministering BCG to said subject.
 143. The method of claim 142, whereinsaid BCG is administered in a unit dosage form comprising between about5×10⁵ and about 1×10⁷ colony forming units (cfu) per 0.1 milligrams ofBCG.
 144. The method of claim 143, wherein said unit dosage formcomprises between about 1×10⁶ and about 6×10⁶ cfu per 0.1 milligrams ofBCG.
 145. The method of claim 144, wherein said unit dosage formcomprises between about 1.8×10⁶ and about 3.9×10⁶ cfu per 0.1 milligramsof BCG.
 146. The method of any one of claims 142-145, wherein said BCGis administered to said subject about once every 1-20 years.
 147. Themethod of claim 146, wherein said BCG is administered to said subjectabout once every 1-10 years.
 148. The method of claim 147, wherein saidBCG is administered to said subject about once every 5 years.
 149. Themethod of any one of claims 146-148, wherein said subject isadministered a total of 1-20 doses of said BCG.
 150. The method of claim149, wherein said subject is administered a total of 2-5 doses of saidBCG.
 151. The method of claim 150, wherein said subject is administereda total of 2 doses of said BCG.
 152. The method of any one of claims87-103 and 104-140, wherein said therapeutic agent is a hypolipidemicagent selected from the group consisting of a HMG-CoA reductaseinhibitor, niacin, a fibric acid derivative, a cholesterol absorptioninhibitor, and a lipolytic agent.
 153. The method of claim 152, saidmethod further comprising administering to said subject a hypolipidemicagent selected from the group consisting of a HMG-CoA reductaseinhibitor, niacin, a fibric acid derivative, a cholesterol absorptioninhibitor, and a lipolytic agent.
 154. The method of claim 152 or 153,wherein said hypolipidemic agent is selected from the group consistingof a HMG-CoA reductase inhibitor, niacin, a fibric acid derivative, acholesterol absorption inhibitor, and a lipolytic agent.
 155. The methodof claim 154, wherein said HMG-CoA reductase inhibitor is selected fromthe group consisting of atorvastatin, cerivastatin, fluvastatin,lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin,simvastatin, and combinations thereof.
 156. The method of claim 155,wherein said fibric acid derivative is selected from the groupconsisting of fenofibrate and gemfibrozil.
 157. The method of claim 155,wherein said cholesterol absorption inhibitor is ezetimibe.
 158. Themethod of claim 155, wherein said lipolytic agent is selected from thegroup consisting of norepinephrine, isoproterenol, forskolin,bucladesine, and theophylline.
 159. The method of any one of claims142-158, said method further comprising administering an additionaltherapeutic agent to said subject.
 160. The method of claim 159, whereinsaid additional therapeutic agent is selected from the group consistingof TNFα, a TNFR2 agonist, and an immunotherapy agent.
 161. The method ofclaim 160, wherein said immunotherapy agent is selected from the groupconsisting of an anti-CTLA-4 agent, an anti-PD-1 agent, an anti-PD-L1agent, an anti-PD-L2 agent, a TNFα cross-linking agent, a TRAILcross-linking agent, a CD27 agent, a CD30 agent, a CD40 agent, a 4-1 BBagent, a GITR agent, an OX40 agent, a TRAILR1 agent, a TRAILR2 agent,and a TWEAKR agent.
 162. The method of any one of claims 1-161, whereinsaid subject is a mammal.
 163. The method of claim 162, wherein saidmammal is a human.
 164. A kit comprising BCG and a package insertinstructing a user of said kit to treat a subject according to themethod of any one of claims 1-85.
 165. The kit of claim 164, wherein kitfurther comprises the additional therapeutic agent of any one of claims159-161.
 166. A kit comprising an agent that can be used to determinethe methylation state of one or more cytosine residues, wherein said kitfurther comprises a package insert instructing a user of said kit toperform the method of any one of claims 86-103.
 167. A kit comprising anagent that can be used to detect one or more mRNA molecules, whereinsaid kit further comprises a package insert instructing a user of saidkit to perform the method of any one of claims 104-106, 107, 109, 110,113, and 115-119.
 168. A kit comprising an agent that can be used todetect one or more proteins, wherein said kit further comprises apackage insert instructing a user of said kit to perform the method ofany one of claims 104-106, 108-110, and 114-119.
 169. A method ofreducing the level of glucose in the blood of a subject in need thereofcomprising administering BCG to said subject.
 170. The method of claim169, whereby said administering lowers the level of glucose in the bloodof said subject by about 0.1% or more relative to a measurement of bloodglucose in said subject prior to administration of said BCG.
 171. Themethod of claim 170, whereby said administering lowers the level ofglucose in the blood of said subject by from about 10% to about 40%relative to a measurement of blood glucose in said subject prior toadministration of said BCG.
 172. The method of claim 169, whereby saidadministering lowers the level of glucose in the blood of said subjectby about 5% or more relative to a control subject not treated with saidBCG, preferably wherein said control subject has the same age and/orgender as said subject.
 173. The method of claim 172, whereby saidadministering lowers the level of glucose in the blood of said subjectby from about 10% to about 40% relative to a control subject not treatedwith said BCG, preferably wherein said control subject has the same ageand/or gender as said subject.
 174. The method of any one of claims169-173, whereby said administering lowers the level of glucose in theblood of said subject within about 1 week to about 7 weeks after beingtreated with said BCG.
 175. The method of any one of claims 169-174,whereby said administering lowers the level of glycated hemoglobin inthe blood of said subject.
 176. The method of claim 175, whereby saidadministering lowers the level of glycated hemoglobin in the blood ofsaid subject by about 5% or more relative to a measurement of glycatedhemoglobin in said subject prior to administration of said BCG.
 177. Themethod of claim 176, whereby said administering lowers the level ofglycated hemoglobin in the blood of said subject by about 15% or morerelative to a measurement of glycated hemoglobin in said subject priorto administration of said BCG.
 178. The method of claim 175, wherebysaid administering lowers the level of glycated hemoglobin in the bloodof said subject by about 5% or more relative to a subject not treatedwith said BCG, preferably wherein said control subject has the same ageand/or gender as said subject.
 179. The method of claim 178, wherebysaid administering lowers the level of glycated hemoglobin in the bloodof said subject by about 15% or more relative to a subject not treatedwith said BCG, preferably wherein said control subject has the same ageand/or gender as said subject.
 180. The method of any one of claims173-179, whereby said administering lowers the level of glycatedhemoglobin in the blood of said subject within about 2 weeks to about 8years after being treated with said BCG.
 181. The method of any one ofclaims 169-180, whereby said administering causes an increase in therate of glycolysis in said subject relative to the rate of glycolysis inthe subject prior to administration of said BCG.
 182. The method of anyone of claims 169-181, whereby said administering causes an increase influx through the pentose phosphate shunt in said subject relative toflux through the pentose phosphate shunt in the subject prior toadministration of said BCG.
 183. The method of any one of claims169-182, whereby said administering causes a decrease in the rate ofoxidative phosphorylation of adenosine diphosphate in said subjectrelative to the rate of oxidative phosphorylation of adenosinediphosphate in the subject prior to administration of said BCG.
 184. Themethod of any one of claims 169-183, whereby said administeringincreases the level of lactate in the blood of said subject relative toa measurement of lactate in the blood of the subject prior toadministration of said BCG.
 185. The method of any one of claims169-184, whereby said administering increases the level of1,5-anhydroglucitol in the blood of said subject relative to ameasurement of 1,5-anhydroglucitol in the blood of the subject prior toadministration of said BCG.
 186. The method of any one of claims169-185, whereby said administering lowers the level of α-ketobutyratein the blood of said subject relative to a measurement of α-ketobutyratein the blood of the subject prior to administration of said BCG. 187.The method of any one of claims 169-186, whereby said administeringlowers the level of 2-hydroxybutyrate in the blood of said subjectrelative to a measurement of 2-hydroxybutyrate in the blood of thesubject prior to administration of said BCG.
 188. The method of any oneof claims 169-187, whereby said administering increases the expressionof hypoxia-inducible factor 1-α (HIF1-α) in the blood of said subjectrelative to a measurement of HIF1-α in the blood of the subject prior toadministration of said BCG.
 189. The method of claim 188, whereby saidadministering increases the expression of HIF1-α in a lymphocyte in saidsubject.
 190. The method of claim 189, wherein said lymphocyte is aperipheral blood lymphocyte.
 191. The method of claim 188, whereby saidadministering increases the expression of HIF1-α in a monocyte in saidsubject.
 192. The method of any one of claims 188-191, wherein saidexpression of HIF1-α is assessed by monitoring HIF1-α mRNA in a sampleisolated from said subject.
 193. The method of any one of claims188-192, whereby said administering increases expression of HIF-1α mRNAby from about 3-fold to about 6-fold.
 194. The method of any one ofclaims 169-193, whereby said administering increases the expression of aglucose transporter in the blood of said subject relative to ameasurement of said glucose transporter in the blood of the subjectprior to administration of said BCG.
 195. The method of claim 194,wherein said glucose transporter is solute carrier family 2 member 6(SLC2A6).
 196. The method of claim 194 or 195, wherein said expressionof said glucose transporter is assessed by monitoring mRNA encoding saidglucose transporter in a sample isolated from said subject.
 197. Themethod of any one of claims 169-196, whereby said administeringincreases the expression of a glycolytic enzyme in the blood of saidsubject relative to a measurement of said glycolytic enzyme in the bloodof the subject prior to administration of said BCG.
 198. The method ofclaim 197, wherein said glycolytic enzyme is selected from the groupconsisting of hexokinase 2 (HK2), glucose-6-phosphate isomerase (G6PI),triosephosphate isomerase 1 (TPI1), galactokinase 1 (GALK1), andgalactose mutarotase (GALM).
 199. The method of claim 197 or 198,wherein said expression of said glycolytic enzyme is assessed bymonitoring mRNA encoding said glycolytic enzyme in a sample isolatedfrom said subject.
 200. The method of any one of claims 169-199, wherebysaid administering reduces the expression of an enzyme that promotesflux through the Krebs cycle in the blood of said subject relative to ameasurement of said enzyme that promotes flux through the Krebs cycle inthe blood of the subject prior to administration of said BCG.
 201. Themethod of claim 200, wherein said enzyme that promotes flux through theKrebs cycle is selected from the group consisting of adenosinetriphosphate citrate lyase (ACLY), aconitase 2 (ACO2), citrate synthase(CS), dihydrolipoamide dehydrogenase (DLD), oxoglutarate dehydrogenase(OGDH), succinate dehydrogenase iron-sulfur complex subunit B (SDHB),and succinate-CoA ligase subunit α (SUCLG1).
 202. The method of claim200 or 201, wherein said expression of said enzyme that promotes fluxthrough the Krebs cycle is assessed by monitoring mRNA encoding saidglycolytic enzyme in a sample isolated from said subject.
 203. Themethod of any one of claims 169-202, wherein said subject has a bloodglucose level of about 100 mg/dL or greater, preferably wherein saidsubject has a blood glucose level of about 126 mg/dL or greater. 204.The method of claim 203, wherein said subject has a blood glucose levelof about 200 mg/dL or greater.
 205. The method of any one of claims169-204, wherein said subject has a blood glucose level that is higherthan that which has previously been observed for said subject.
 206. Themethod of claim 205, wherein said subject has previously been observedas having a blood glucose level of less than about 200 mg/dL.
 207. Themethod of claim 206, wherein said subject has previously been observedas having a blood glucose level of less than about 126 mg/dL.
 208. Themethod of claim 207, wherein said subject has previously been observedas having a blood glucose level of about 100 mg/dL or less.
 209. Themethod of any one of claims 169-208, wherein said subject is sufferingfrom a disease associated with an elevated blood glucose level.
 210. Themethod of claim 209, wherein said disease is selected from the groupconsisting of type 2 diabetes, noninsulin-dependent diabetes mellitus(NIDDM), nonalcoholic steatohepatitis (NASH), metabolic syndrome, cysticfibrosis, drug induced hyperglycemia, insulin resistance syndromes,diseases caused by genetic mutations in the pancreas, cancer, infection,Leprechaunism, Rabson Mandenhall syndrome, lipoatrophic diabetes,pancreatitis, trauma, hemochromatoisis, fibrocalculous pancreatopathy,acromegaly, Cushings syndrome, glucagonoma, pheochromocytoma,hyperthyroism, somatostatinoma, aldosteroma, infections associated withbeta cell destruction, Rubella, coxsachie virus B, mumps,cytomegatolovirus infection, adenovirus infection, a genetic syndrome,stiff person syndrome, anti-insulin receptor abnormalities, liverdisease, and renal failure.
 211. The method of claim 210, wherein saiddrug induced hyperglycemia is induced by one or more agents selectedfrom the group consisting of steroids, cortisol, thiazides, diazocide,calcineurin inhibitors, oral contraceptives, beta adrenergic agonists,nicotinic acid, pentamidine, alpha interferon, anti-psychotic agents,anti-retroviral agents, and rodenticides.
 212. The method of claim 211,wherein said rodenticide is pyrinuron.
 213. The method of claim 210,wherein said cancer is pancreatic cancer.
 214. The method of claim 210,wherein said genetic syndrome is selected from the group consisting ofDown's syndrome, Klinefelter's syndrome, Turner syndrome, Woldframsyndrome, and Friendreich ataxia.
 215. The method of any one of claims169-214, wherein said subject has undergone a pancreatectomy.
 216. Themethod of any one of claims 169-215, wherein said subject exhibits oneor more mutations in a mitochondrial gene.
 217. The method of any one ofclaims 169-216, wherein said subject exhibits one or more mutations in agene selected from the group consisting of hepatic nuclear factor 1(MODY3), glucokinase (MODY2), and hepatocyte nuclear factor 4-α (MODY1).218. The method of any one of claims 169-217, wherein said BCG isadministered to said subject in an amount sufficient to alleviate orreduce a symptom associated with said disease.
 219. The method of claim218, wherein said symptom is polyphagia, polydipsia, polyuria, blurredvision, fatigue, cardiac arrhythmia, stupor, dry mouth, and poor woundhealing.
 220. The method of any one of claims 169-219, wherein said BCGis administered in a unit dosage form comprising between about 5×10⁵ andabout 1×10⁷ cfu per 0.1 milligrams of BCG.
 221. The method of claim 220,wherein said unit dosage form comprises between about 1×10⁶ and about6×10⁶ cfu per 0.1 milligrams of BCG.
 222. The method of claim 221,wherein said unit dosage form comprises between about 1.8×10⁶ and about3.9×10⁶ cfu per 0.1 milligrams of BCG.
 223. The method of any one ofclaims 169-222, wherein said BCG is administered to said subject aboutonce every 1-20 years.
 224. The method of claim 223, wherein said BCG isadministered to said subject about once every 1-10 years.
 225. Themethod of claim 224, wherein said BCG is administered to said subjectabout once every 5 years.
 226. The method of any one of claims 169-225,wherein said subject is administered a total of 1-20 doses of said BCG.227. The method of claim 226, wherein said subject is administered atotal of 2-5 doses of said BCG.
 228. The method of claim 227, whereinsaid subject is administered a total of 2 doses of said BCG.
 229. Themethod of claim 228, wherein said subject is administered a first doseof BCG followed by a second dose of BCG from about 2 weeks to about 8weeks after administration of said first dose.
 230. The method of claim229, wherein said subject is administered a first dose of BCG followedby a second dose of BCG about 4 weeks after administration of said firstdose.
 231. The method of any one of claims 1-85, 142-151, 159-163, and169-230, wherein administration of said BCG increases the expression ofnuclear receptor subfamily 1 group H member 3 (NR1H3).
 232. The methodof claim 231, wherein said expression of NR1H3 is assessed by monitoringNR1H3 mRNA.
 233. The method of any one of claims 1-85, 142-151, 159-163,and 169-232, wherein administration of said BCG increases the expressionof a gene selected from the group consisting of adenosine triphosphatebinding cassette subfamily A member 1 (ABCA1), adenosine triphosphatebinding cassette subfamily G (ABCG), apolipoprotein E(APOE), Fas cellsurface death receptor (FAS), and stearoyl-CoA desaturase (SCD1)relative to a measurement of the expression of said gene in said subjectprior to administration of said BCG.
 234. The method of any one ofclaims 1-85, 142-151, 159-163, and 169-233, wherein administration ofsaid BCG reduces the expression of a gene selected from the groupconsisting of fructose-bisphosphatase 1 (FBP1), glucose-6-phosphatedehydrogenase (G6PD), and muscle pyruvate kinase (PKM) relative to ameasurement of the expression of said gene in said subject prior toadministration of said BCG.
 235. The method of any one of claims169-234, wherein said subject is not suffering from a disease orcondition selected from the group consisting of an autoimmune disease, aneurological condition, an allergy, allograft rejection,graft-versus-host disease, asthma, macular degeneration, muscularatrophy, a disease related to miscarriage, atherosclerosis, bone loss, amusculoskeletal disease, and obesity.
 236. The method of any one ofclaims 169-234, wherein said subject is not suffering from an autoimmunedisease.
 237. The method of any one of claims 169-234, wherein saidsubject is not suffering from an autoimmune disease selected from thegroup consisting of type I diabetes, Alopecia Areata, AnkylosingSpondylitis, Antiphospholipid Syndrome, Autoimmune Addison's Disease,Autoimmune Hemolytic Anemia, Autoimmune Hepatitis, Behcet's Disease,Bullous Pemphigoid, Cardiomyopathy, Celiac Sprue-Dermatitis, ChronicFatigue Immune Dysfunction Syndrome (CFIDS), Chronic InflammatoryDemyelinating Polyneuropathy, Churg-Strauss Syndrome, CicatricialPemphigoid, CREST Syndrome, Cold Agglutinin Disease, Crohn's Disease,Essential Mixed Cryoglobulinemia, Fibromyalgia-Fibromyositis, Graves'Disease, Guillain-Barré, Hashimoto's Thyroiditis, Hypothyroidism,Idiopathic Pulmonary Fibrosis, Idiopathic Thrombocytopenia Purpura(ITP), IgA Nephropathy, Juvenile Arthritis, Lichen Planus, Lupus,Ménière's Disease, Mixed Connective Tissue Disease, Multiple Sclerosis,Myasthenia Gravis, Pemphigus Vulgaris, Pernicious Anemia, PolyarteritisNodosa, Polychondritis, Polyglandular Syndromes, Polymyalgia Rheumatica,Polymyositis and Dermatomyositis, Primary Agammaglobulinemia, PrimaryBiliary Cirrhosis, Psoriasis, Raynaud's Phenomenon, Reiter's Syndrome,Rheumatic Fever, Rheumatoid Arthritis, Sarcoidosis, Scleroderma,Sjögren's Syndrome, Stiff-Man Syndrome, Takayasu Arteritis, TemporalArteritis/Giant Cell Arteritis, Ulcerative Colitis, Uveitis, Vasculitis,Vitiligo, and Wegener's Granulomatosis.
 238. The method of any one ofclaims 169-234, wherein said subject is not suffering from aneurological condition.
 239. The method of any one of claims 169-234,wherein said subject is not suffering from a neurological conditionselected from the group consisting of a brain tumor, a brain metastasis,a spinal cord injury, schizophrenia, epilepsy, Amyotrophic lateralsclerosis (ALS), Parkinson's disease, Autism, Alzheimer's disease,Huntington's disease, and stroke.
 240. The method of any one of claims169-234, wherein said subject is not suffering from an allergy.
 241. Themethod of any one of claims 169-234, wherein said subject is notsuffering from an allergy selected from the group consisting of foodallergy, seasonal allergy, pet allergy, hives, hay fever, allergicconjunctivitis, poison ivy allergy oak allergy, mold allergy, drugallergy, dust allergy, cosmetic allergy, and chemical allergy.
 242. Amethod of diagnosing a subject as having hyperglycemia or a diseaseassociated therewith, said method comprising: a) i) determining a levelof one or more substances selected from the group consisting of glucose,cholesterol, LDL, a triglyceride, glycated hemoglobin, an mRNA moleculeencoding a protein selected from the group consisting of FBP1, G6PD, andPKM, a protein selected from the group consisting of FBP1, G6PD, andPKM, an mRNA molecule encoding an enzyme that promotes flux through theKrebs cycle, an enzyme that promotes flux through the Krebs cycle,α-ketobutyrate, and 2-hydroxybutyrate in a sample from said subject; and(ii) comparing the level of the one or more substances to the level ofthe one or more substances in a reference sample, wherein adetermination that the level of said one or more substances in thesample from the subject is greater than the level of said one or moresubstances in the reference sample indicates that the subject has saiddisease; or b) (i) determining a level of one or more substancesselected from the group consisting of an mRNA molecule encoding aglycolytic enzyme, a glycolytic enzyme, an mRNA molecule encoding aglucose transporter, a glucose transporter, an mRNA molecule encodingHIF1-α, HIF1-α, an mRNA molecule encoding NR1H3, NR1H3, lactate, and1,5-anhydroglucitol in a sample from said subject; and (ii) comparingthe level of the one or more substances to the level of the one or moresubstances in a reference sample, wherein a determination that the levelof said one or more substances in the sample from the subject is lessthan the level of said one or more substances in the reference sampleindicates that the subject has said disease.
 243. The method of claim242, said method further comprising determining whether the subject islikely to respond to treatment with a therapeutic agent for saiddisease, wherein a determination that the level of the one or moresubstances listed in (a) in the sample from the subject having saidhyperglycemia or disease associated therewith is greater than the levelof said one or more substances in the reference sample indicates thatthe subject is likely to respond to said treatment, or wherein adetermination that the level of the one or more substances listed in (b)in the sample from the subject having said disease is less than thelevel of said one or more substances in the reference sample indicatesthat the subject is likely to respond to said treatment.
 244. A methodof determining whether a subject previously administered a therapeuticagent for the treatment of a disease would benefit from receiving one ormore additional doses of said therapeutic agent, said method comprising:a) (i) determining a level of one or more substances selected from thegroup consisting of glucose, cholesterol, LDL, a triglyceride, glycatedhemoglobin, an mRNA molecule encoding a protein selected from the groupconsisting of FBP1, G6PD, and PKM, a protein selected from the groupconsisting of FBP1, G6PD, and PKM, an mRNA molecule encoding an enzymethat promotes flux through the Krebs cycle, an enzyme that promotes fluxthrough the Krebs cycle, α-ketobutyrate, and 2-hydroxybutyrate in asample from said subject; and (ii) comparing the level of the one ormore substances to the level of the one or more substances in areference sample, wherein a determination that the level of said one ormore substances in the sample from the subject is the same as or greaterthan the level of said one or more substances in said reference sampleindicates that said subject would benefit from one or more additionaldoses of said therapeutic agent, or b) (i) determining a level of one ormore substances selected from the group consisting of an mRNA moleculeencoding a glycolytic enzyme, a glycolytic enzyme, an mRNA moleculeencoding a glucose transporter, a glucose transporter, an mRNA moleculeencoding HIF1-α, HIF1-α, an mRNA molecule encoding NR1H3, NR1H3,lactate, and 1,5-anhydroglucitol in a sample from said subject; and (ii)comparing the level of the one or more substances to the level of theone or more substances in a reference sample, wherein a determinationthat the level of said one or more substances in the sample from thesubject is the same as or less than the level of said one or moresubstances in said reference sample indicates that said subject wouldbenefit from one or more additional doses of said therapeutic agent.245. The method of any one of claims 242-244, wherein said enzyme thatpromotes flux through the Krebs cycle is selected from the groupconsisting of ACLY, ACO2, CS, DLD, OGDH, SDHB, and SUCLG1.
 246. Themethod of any one of claims 242-244, wherein said glycolytic enzyme isselected from the group consisting of HK2, G6PI, TPI1, GALK1, and GALM.247. The method of any one of claims 242-244, wherein said glucosetransporter is SLC2A6.
 248. The method of any one of claims 242-247,wherein the level of said mRNA molecule is determined by performing anassay selected from the group consisting of reverse transcription PCR(RT-PCR) and a Northern blot.
 249. The method of any one of claims242-247, wherein the level of said enzyme that promotes flux through theKrebs cycle, glycolytic enzyme, glucose transporter, HIF-1a, NR1H3,FBP1, G6PD, or PKM is determined by performing an assay selected fromthe group consisting of an immunoblot and an enzyme-linked immunosorbantassay (ELISA).
 250. The method of any one of claims 242-247, wherein thelevel of said α-ketobutyrate, 2-hydroxybutyrate, lactate, or1,5-anhydroglucitol is determined by nuclear magnetic resonance (NMR)spectroscopy.
 251. The method of claim 242 or 243, wherein saidreference sample is a sample isolated from a control subject not havingsaid disease.
 252. The method of claim 251, wherein said control subjectis a subject of the same age and/or gender of said subject having saiddisease.
 253. The method of claim 244, wherein said reference sample isa prior sample that has been previously isolated from said subject. 254.The method of claim 253, wherein said prior sample was isolated betweenabout 24 hours and about 5 years before making said determination. 255.The method of claim 254, wherein said prior sample was isolated betweenabout 1 month and about 1 year prior to making said determination. 256.The method of any one of claims 253-255, wherein said prior sample wasisolated prior to said subject being administered said therapeuticagent.
 257. The method of any one of claims 242-256, wherein saiddisease associated with hyperglycemia is selected from the groupconsisting of type 2 diabetes, noninsulin-dependent diabetes mellitus(NIDDM), nonalcoholic steatohepatitis (NASH), metabolic syndrome, cysticfibrosis, drug induced hyperglycemia, insulin resistance syndromes,diseases caused by genetic mutations in the pancreas, cancer, infection,Leprechaunism, Rabson Mandenhall syndrome, lipoatrophic diabetes,pancreatitis, trauma, hemochromatoisis, fibrocalculous pancreatopathy,acromegaly, Cushings syndrome, glucagonoma, pheochromocytoma,hyperthyroism, somatostatinoma, aldosteroma, infections associated withbeta cell destruction, Rubella, coxsachie virus B, mumps,cytomegatolovirus infection, adenovirus infection, a genetic syndrome,stiff person syndrome, anti-insulin receptor abnormalities, liverdisease, and renal failure.
 258. The method of claim 257, wherein saiddrug induced hyperglycemia is induced by one or more agents selectedfrom the group consisting of steroids, cortisol, thiazides, diazocide,calcineurin inhibitors, oral contraceptives, beta adrenergic agonists,nicotinic acid, pentamidine, alpha interferon, anti-psychotic agents,anti-retroviral agents, and rodenticides.
 259. The method of claim 258,wherein said rodenticide is pyrinuron.
 260. The method of claim 257,wherein said cancer is pancreatic cancer.
 261. The method of claim 257,wherein said genetic syndrome is selected from the group consisting ofDown's syndrome, Klinefelter's syndrome, Turner syndrome, Woldframsyndrome, and Friendreich ataxia.
 262. The method of any one of claims244-250 and 253-261, said method comprising administering saidtherapeutic agent to said subject identified as a subject that wouldbenefit from one or more additional doses of said therapeutic agent.263. The method of any one of claims 244-250 and 253-262, wherein saidtherapeutic agent is BCG.
 264. The method of any one of claims 242-252and 257-261, said method comprising administering BCG to said subjectidentified as having hyperglycemia or a disease associated therewith.265. The method of any one of claims 169-241 and 262-264, wherein BCG isthe only therapeutic agent administered to said subject.
 266. The methodof any one of claims 169-241 and 262-264, wherein said subject is notadministered an agent that promotes the expression of IL-2.
 267. Themethod of any one of claims 169-241 and 262-264, wherein said subject isnot administered lymphotoxin.
 268. The method of any one of claims169-241 and 262-264, wherein said subject is not administered Lentinan.269. The method of any one of claims 169-241 and 262-264, wherein saidsubject is not administered an agent that promotes the expression ofTNF-α other than BCG.
 270. The method of any one of claims 169-268,wherein said subject is a mammal.
 271. The method of claim 269, whereinsaid mammal is a human.
 272. A kit comprising BCG and a package insertinstructing a user of said kit to perform the method of any one ofclaims 169-270.
 273. The method of claim 169, wherein said disease isselected from the group consisting of type 2 diabetes,noninsulin-dependent diabetes mellitus (NIDDM), nonalcoholicsteatohepatitis (NASH), metabolic syndrome, cystic fibrosis, druginduced hyperglycemia, insulin resistance syndromes, diseases caused bygenetic mutations in the pancreas, cancer, infection, Leprechaunism,Rabson Mandenhall syndrome, lipoatrophic diabetes, pancreatitis, trauma,hemochromatoisis, fibrocalculous pancreatopathy, acromegaly, Cushingssyndrome, glucagonoma, pheochromocytoma, hyperthyroism, somatostatinoma,aldosteroma, infections associated with beta cell destruction, Rubella,coxsachie virus B, mumps, cytomegatolovirus infection, adenovirusinfection, a genetic syndrome, stiff person syndrome, anti-insulinreceptor abnormalities, liver disease, and renal failure.
 274. Themethod of claim 169, wherein said BCG is administered in a unit dosageform comprising between about 5×10⁵ and about 1×10⁷ cfu per 0.1milligrams of BCG.
 275. The method of claim 274, wherein said unitdosage form comprises between about 1×10⁶ and about 6×10⁶ cfu per 0.1milligrams of BCG.
 276. The method of claim 169, wherein said subject isnot suffering from a disease or condition selected from the groupconsisting of an autoimmune disease, a neurological condition, anallergy, allograft rejection, graft-versus-host disease, asthma, maculardegeneration, muscular atrophy, a disease related to miscarriage,atherosclerosis, bone loss, a musculoskeletal disease, and obesity. 277.The method of claim 169, wherein said subject is not suffering from anautoimmune disease.
 278. The method of claim 169, wherein said subjectis not suffering from an autoimmune disease selected from the groupconsisting of type I diabetes, Alopecia Areata, Ankylosing Spondylitis,Antiphospholipid Syndrome, Autoimmune Addison's Disease, AutoimmuneHemolytic Anemia, Autoimmune Hepatitis, Behcet's Disease, BullousPemphigoid, Cardiomyopathy, Celiac Sprue-Dermatitis, Chronic FatigueImmune Dysfunction Syndrome (CFIDS), Chronic Inflammatory DemyelinatingPolyneuropathy, Churg-Strauss Syndrome, Cicatricial Pemphigoid, CRESTSyndrome, Cold Agglutinin Disease, Crohn's Disease, Essential MixedCryoglobulinemia, Fibromyalgia-Fibromyositis, Graves' Disease,Guillain-Barré, Hashimoto's Thyroiditis, Hypothyroidism, IdiopathicPulmonary Fibrosis, Idiopathic Thrombocytopenia Purpura (ITP), IgANephropathy, Juvenile Arthritis, Lichen Planus, Lupus, Ménière'sDisease, Mixed Connective Tissue Disease, Multiple Sclerosis, MyastheniaGravis, Pemphigus Vulgaris, Pernicious Anemia, Polyarteritis Nodosa,Polychondritis, Polyglandular Syndromes, Polymyalgia Rheumatica,Polymyositis and Dermatomyositis, Primary Agammaglobulinemia, PrimaryBiliary Cirrhosis, Psoriasis, Raynaud's Phenomenon, Reiter's Syndrome,Rheumatic Fever, Rheumatoid Arthritis, Sarcoidosis, Scleroderma,Sjögren's Syndrome, Stiff-Man Syndrome, Takayasu Arteritis, TemporalArteritis/Giant Cell Arteritis, Ulcerative Colitis, Uveitis, Vasculitis,Vitiligo, and Wegener's Granulomatosis.
 279. The method of claim 169,wherein said subject is not suffering from type I diabetes.
 280. Themethod of claim 169, wherein said subject is a human.
 281. A method ofinducing an increase in the rate of aerobic glycolysis in a mammaliansubject, said method comprising administering to said subject BCG. 282.The method of claim 281, wherein the rate of aerobic glycolysis isincreased relative to a measurement of aerobic glycolysis in saidsubject prior to administration of said BCG.
 283. The method of claim281, whereby said administering reduces the rate of oxidativephosphorylation of adenosine diphosphate in said subject relative to ameasurement of oxidative phosphorylation of adenosine diphosphate insaid subject prior to administration of said BCG.
 284. The method ofclaim 281, whereby said administering reduces the level of one or moresubstances selected from the group consisting of glucose, cholesterol,LDL, a triglyceride, glycated hemoglobin, an mRNA molecule encoding aprotein selected from the group consisting of FBP1, G6PD, and PKM, aprotein selected from the group consisting of FBP1, G6PD, and PKM, anmRNA molecule encoding an enzyme that promotes flux through the Krebscycle, an enzyme that promotes flux through the Krebs cycle,α-ketobutyrate, and 2-hydroxybutyrate in said subject relative to ameasure of said substance in said subject prior to administration ofsaid BCG.
 285. The method of claim 281, whereby said administeringincreases the level of one or more substances selected from the groupconsisting of an mRNA molecule encoding a glycolytic enzyme, aglycolytic enzyme, an mRNA molecule encoding a glucose transporter, aglucose transporter, an mRNA molecule encoding HIF1-α, HIF1-α, an mRNAmolecule encoding NR1H3, NR1H3, lactate, and 1,5-anhydroglucitol in saidsubject relative to a measure of said substance in said subject prior toadministration of said BCG.
 286. The method of claim 284, wherein saidenzyme that promotes flux through the Krebs cycle is selected from thegroup consisting of ACLY, ACO2, CS, DLD, OGDH, SDHB, and SUCLG1. 287.The method of claim 285, wherein said glycolytic enzyme is selected fromthe group consisting of HK2, G6PI, TPI1, GALK1, and GALM.
 288. Themethod of claim 285, wherein said glucose transporter is SLC2A6. 289.The method of claim 281, wherein said mammalian subject is a human.